Dosing regime and formulations for type B adenovirus

ABSTRACT

A method of treating a human patient comprising systemically administering multiple doses of a parenteral formulation of a replication capable oncolytic adenovirus of subgroup B in a single treatment cycle, wherein the total dose given in each dose is in the range of 1×10 10  to 1×10 14  viral particles, and wherein each dose of virus is administered over a period of 1 to 90 minutes, for example at a rate of viral particle delivery in the range of 2×10 10  particles per minute to 2×10 12  particles per minute. Also provided are formulations of the oncolytic adenoviruses and combination therapies of the viruses and formulations with other therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a US national phase of International Application No.PCT/EP2014/062284 filed on Jun. 12, 2014, which claims priority to GreatBritain Patent Application No. 1310698.4 filed on Jun. 14, 2013; GreatBritain Patent Application No. 1405140.3 filed on Mar. 22, 2014; andGreat Britain Patent Application No. 1406509.8 filed on Apr. 10, 2014.The entire disclosure contents of each of these applications are herebyincorporated by reference into the present application.

The present disclosure relates to a method of treating a patient, forexample with a replication capable oncolytic adenovirus employing adosing regimen designed to allow the virus to have a suitabletherapeutic effect and/or minimise adverse events in vivo. Thedisclosure also extends to formulations described herein, methods ofpreparing said formulations and use of the same in treatment, inparticular the treatment of cancer.

BACKGROUND

Cancer is a leading cause of death and serious illness worldwide. Thereare over 200 different types of cancer and the type of treatment isdependent on the type of cancer. Typically, treatment will involvesurgery, chemotherapy and/or radiotherapy. These treatments are oftenunsuccessful or are only partially successful and have significant sideeffects. Five year survival rates for cancer can range from less than 5%to over 95% depending on the type of cancer (CRUK statistics,2000-2001). For example, between 2005-2009, patients with colorectalcancer, which accounts for 13% of all cancers in men and women in theUK, had a five year survival rate of approximately 55% in the UK. Thisdrops to just 12% for patients with metastatic colorectal cancer.

The management of metastatic cancer is mainly palliative and involves acombination of palliative surgery, chemotherapy, radiation andsupportive care. Clinical outcomes such as overall survival, responseand toxicity are important, but alternative outcomes such asprogression-free survival, quality of life, convenience, acceptabilityand patient choice are also important. New therapies are clearly neededto improve these outcomes.

During transformation, cancer cells acquire certain mutations whichrender them more permissive to virus infection. Cancer cells also inducethe suppression of host anti-tumour activity. Changes within the tumourcells and the local micro-environment create a potential vulnerabilityand expose the tumour to infection by viruses (Liu et al 2007; Liu et al2008;

Roberts, 2006).

There is a long history of using viruses to treat cancer beginning withanecdotal reports of temporary cancer remission after natural viralinfections or viral vaccinations. The earliest report seems to be a 1912account of the regression of cervical cancer in a patient vaccinated forrabies. Similar results were seen in cancer patients receiving smallpoxvaccinations, or following natural virus infections such as mumps ormeasles. Based on these reports as well as animal data, inoculations oflive viruses into patients for cancer treatment were initiated in thelate 1940s and early 1950s.

The usual experience, however, was that after occasional temporarytumour regression, the tumour regrew and the patient died. Theseinoculations seldom resulted in long-lasting complete remissions. In1957, Albert B. Sabin, M. D., who developed the live oral polio vaccinecommented, “The most disappointing aspect is the fact that even when avirus is oncolytic and it punches a hole in a tumour, the immuneresponse of the individual to the virus occurs so fast that the effectsare quickly wiped out and the tumour continues to grow.”

At the present time, a number of oncolytic viruses have been identifiedbut the only virus that has been approved for clinical use anywhere inthe world to date is Oncorine (H101) a subgroup C adenovirus modified byE1B-55KD deletion enabling conditional replication in P53-deficientcancer cells (H101 is a close analogue of ONYX015 as described byBischoff et al 1996). Oncorine is administered by intratumouralinjection for head and neck cancer.

Talimogene laherparepvec (Tvec), is an oncolytic virus based on Herpessimplex virus type-1 carrying ICP34.5 & ICP47 deletions, expressing US11as an immediate early gene and encoding GM-CSF. The OPTiM trial is amulti-national, open label, randomized study designed to assess theefficacy and safety of treatment with talimogene laherparepvec as anintratumoural treatment compared to subcutaneously administered GM-CSFin patients with unresectable stage Ill (b-c) and Stage IV (M1a-c)disease. On interim analysis, talimogene laherparepvec elicited adurable response rate in 16% of patients compared to 2% in thosereceiving GM-CSF. Other oncolytic viruses for intra-tumouraladministration currently in development include (Sheridan 2013):

-   -   Reolysin, an oncolytic reovirus serotype 3 (Dearing strain)    -   PV701, an oncolytic Newcastle disease virus    -   CG0070, a conditionally replicating adenovirus encoding GM-CSF    -   Pexastimogene devacirepvec (Pexa-Vec, JX-594), a thymidine        kinase-deleted vaccinia virus encoding GM-CSF    -   Cavatak, an unmodified Coxsackievirus A21    -   Seprehvir (HSV1716), a conditionally replicating Herpes simplex        type 1 carrying an ICP34.5 deletion    -   DNX-2401, a conditionally replicating adenovirus encoding an        integrin-binding peptide    -   CGTG-102, a conditionally replicating adenovirus encoding GM-CSF

ColoAd1 is a chimeric (Ad11/Ad3) serogroup B adenovirus, which wasdeveloped using the process of directed evolution and it is thought tobe suitable for the treatment of cancers of epithelial origin andmetastatic forms thereof, including colorectal cancer (Kuhn, I et al.2008). To date, clinical studies of oncolytic viruses have primarilyinvestigated intra-tumoural injection of the virus. In a review ofclinical studies by Aghi & Martuza (2005) 25 of 36 studies usedintra-tumoural injection to administer the virus. However, this methodis only practical for treating easily accessible tumours and in patientswhere the structure of the tumour, such as tissue stroma and necroticareas therein, do not limit spread of the virus within a tumour (Ries &Korn 2002).

Death from cancer is often the result of inaccessible tumours ormetastases. Oncolytic viruses administered intra-tumourally rely onsystemic dissemination from the tumour to reach these secondary tumours.However, dissemination has proved transient and often ineffective(Ferguson et al 2012).

Thus, intra-tumoural injection is only suitable for a limited number ofcancers and is not suitable for treatment of, for example of manymetastatic cancers.

Where intravenous administration of oncolytic viruses has been employedgenerally it has been associated with acute toxicity and rapidclearance. For example, in the case of the group C adenovirus Ad5, forwhich uptake is mediated by the ubiquitous coxsackie adenovirus receptor(CAR), side effects including acute liver toxicity, influenza likeillness and haematological changes have been regularly reported, whilstrapid hepatic clearance and immunological neutralisation are also welldescribed.

The currently established view is that repeated doses are required inorder to produce and maintain efficacy. In all the oncolytic cancertreatments under investigation it is generally envisaged that thetreatment will be chronic, with repeat administrations over many weeks,months or years. For example, in the case of PV701, treatment in atleast one patient continued in cycles for about 10 month with 6 daysbetween finishing the previous treatment cycle and initiating the nexttreatment cycle.

Neumanatis et al (2001) report administration of up to 24 weekly cyclesof an intravenous infusion of ONYX-015 to cancer patients. In anon-going phase III clinical study (Clinicaltrials.gov identifierNCT01708993), Reolysin® (an oncloytic reovirus) was infused over a 1hour period on days 1 to 3 and then every 3 weeks until progression. Ina recently reported phase I clinical trial (Clinicaltrials.govidentifier NCT01380600) JX-594 was administered intravenously every 2weeks on four occasions, and in a second on-going phase I/II clinicalstudy (Clinicaltrials.gov identifier NCT01394939) JX-594 is administeredintravenously weekly for 5 weeks followed by up to 3 intra-tumouralboosts to liver metastases of patients with metastatic colorectalcancer. In the on-going OPTIM clinical trial talimogene laherparepvecwas administered intra-tumourally every two weeks for up to 18 months(Clinicaltrials.gov identifier NCT00769704).

SUMMARY OF THE INVENTION

In a first aspect of the disclosure there is provided a method oftreating a human patient, said method comprising the steps of:

-   -   systemically administering multiple doses of a parenteral        formulation of a replication capable oncolytic adenovirus of        subgroup B in a single treatment cycle,    -   wherein the total dose given in each administration is in the        range of 1×10¹⁰ to 1×10¹⁴ viral particles per dose, and

wherein each dose of virus is administered such that the rate of viralparticle delivery is in the range of 2×10¹⁰ particles per minute to2×10¹² particles per minute.

In an independent aspect the present disclosure relates to ColoAd1 foruse in treating ovarian cancer, for example administering atherapeutically effective amount of ColoAd1 to a patient with ovariancancer, for example employing a dosing regimen described herein.

In a further independent aspect the present disclosure relates to acombination therapy comprising oncolytic type B adenovirus, such asColoAd1, and a chemotherapeutic agent which does not interfere with theadenovirus activity, such as viral replication in vivo.

In one embodiment the combination therapy is employed for treatment ofcancer, in particular a cancer described herein, in particularcolorectal cancer or ovarian cancer, including metastatic forms thereof.

In one embodiment ColoAd1 in a combination therapy is dosed according toa regimen described herein.

Also provided is a parenteral formulation of a replication capableoncolytic adenovirus of subgroup B, for use in treatment as describedherein.

The present disclosure also extends to use of a parenteral formulationof a replication capable oncolytic adenovirus of subgroup B, for themanufacture of a medicament, as described herein and for use intreatments described herein.

In one aspect there is also provided a unit dose in the range 1×10¹⁰ to1×10¹⁴, such as 6×10¹² viral particles of a replication capableoncolytic adenovirus of subgroup B.

Also provided is an infusion or injection rate for dosing the viralparticles of 2×10⁹ to 2×10¹² virus particles (VP) per minute, forexample 1.5×10¹¹ VP per minute.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Shows the cytotoxicity profile on A549 cells of ColoAd1 in thepresence of fresh whole human blood.

FIG. 2 Biodistribution of 1e11 (1×10¹¹) particles of ColoAd1 in normalBalbC mice 24 hours post injection.

FIG. 3 Biodistribution of ColoAd1 and ColoAd10132 in CD46 transgenicmice at 1 hr and 72 hrs post-injection.

FIG. 4 Clearance kinetics of ColoAd1 from primary organs liver, spleenand lungs in CD46-expressing mice followed for 65 days.

FIG. 5 Kinetics of ColoAd1 in mice either with or withoutco-administration of neutralising serum.

FIG. 6 Cytokine levels after the first and subsequent equal therapeuticdoses in a pre-clinical toxicology study in CD-1 mice:

FIG. 7A-C Cytokine levels (ng/L) (TNF (A), gamma interferon (B) and IL6(C)) over time in human cancer patients with metastatic solid epithelialtumours after intravenous doses of ColoAd1.

FIG. 8A Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients with metastatic solid epithelialtumours.

FIG. 8B Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients with metastatic solid epithelialtumours.

FIG. 9A Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients administered with 1e10)(1×10¹⁰ ColoAd1viral particles over 5 minutes.

FIG. 9B Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients administered with 1e11 (1×10¹¹) ColoAd1viral particles over 5 minutes.

FIG. 9C Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients administered with 1e12 (1×10¹²) ColoAd1viral particles over 5 minutes.

FIG. 9D Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients administered with 1e13 (1×10¹³) ColoAd1viral particles over 5 minutes.

FIG. 9E Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients administered with 3e12 (3×10¹²) ColoAd1viral particles over 5 minutes.

FIG. 9F Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients administered with 3e12 (3×10¹²) ColoAd1viral particles over 20 minutes.

FIG. 9G Systemic pharmacokinetics of ColoAd1 (Genome copies per mL ofblood) in human cancer patients administered with 6e12 (6×10¹²) ColoAd1viral particles over 40 minutes.

FIG. 10 Slower infusion of the same dose lowers the cMax level at theend of infusion (cohort 5 vs 6)

FIG. 11 MCP1 levels (ng/L) over time in human cancer patients withmetastatic solid epithelial tumours after intravenous doses of ColoAd1.

FIG. 12 Schematic diagram showing ColoAd1 replication cycle in cell.

FIG. 13 ColoAd1 infection of cancer cells shown as nuclear staining in acolorectal cell line following in vitro infection with the virus.

FIG. 14A Nuclear staining (hexon staining) of ColoAd1 in colorectaltissue from a patient with colorectal cancer after administration ofColoAd1 by IT.

FIG. 14B Isotype control staining for FIG. 14A.

FIG. 14C Colorectal tissue (hexon staining) showing no nuclear stainingin stromal cells (following IV administration of ColoAd1 to a colorectalcancer patient.

FIG. 14D Isotype control for FIG. 14C.

FIG. 15 320 compounds (clinically approved or compounds in development)that were analysed for their impact on viral replication.

FIG. 16A-D An in vivo murine model showing the effects of Paclitaxel andColoAd1 combination therapy (and controls).

FIG. 17-18 In vivo data for ColoAd1 and chemotherapy in a murine model.

DETAILED DESCRIPTION

In one embodiment the dose administered is in the range of 1×10¹⁰ to1×10¹³, such as 1×10¹⁰ to 1×10¹² viral particles.

In one embodiment the total dose administered in one treatment cycle is1×10¹², 2×10¹², 3×10¹², 4×10¹², 5×10¹², 6×10¹², 7×10¹², 8×10¹² or 9×10¹²viral particles.

In one embodiment the total dose administered in one treatment cycle is6×10¹² viral particles. It is hypothesised by the present inventors thatwhat may be most critical for efficacy is to establish a productiveinfection within the tumour at an early stage, for example before ananti-viral immune response has developed.

The dosing regimen thus has to balance delivering sufficient virus togenerate, for example adequate plasma levels of virus for a period longenough to seed viral infection in the cancer cells, whilst not elicitingtoxicity and severe adverse events in the patient (or minimising thesame).

The present inventors have shown for the first time that infection ofthe tumour by type B adenovirus can be established by doses of viralparticles administered intravenously. Support for this conclusion isprovided herein where patients with colorectal cancer that receivedtreatment with ColoAd1 intravenously were shown to have virus infectionin the nucleus of cancer cells, when the cells were stained for hexonand also when analysed independently by PCR. Virus in the nucleusindicates the virus life cycle of the virus is in progress and anincrease in the viral load in the patients indicates the virus is ableto replicate.

When administering an oncolytic adenovirus systemically to a patient, anumber of dosing variables need to be considered. These dosing variablesinclude, but are not limited to: the route of virus administration; thedose of virus administered; the rate of viral administration for eachdose; the interval between individual viral administrations in a givencycle; the number of viral administrations per treatment cycle; theinterval between treatment cycles; the number of treatment cycles; andfinally the use of any co-medicaments or other supportive care used toenhance efficacy or minimise adverse effects. Each of the dosingparameters is in turn dependent upon the specific characteristics of thetype of oncolytic virus under investigation.

The key parameters will include, but are not limited to: the relativedegree and avidity of any binding of the virus to tumour cells versusnon-tumour cells; the relative selectivity and potency of the virus intumour cells versus non-tumour cells; the rate of active uptake andclearance of the virus by reticuloendothelial cells (for example liverKupffer cells) and any specific or non-specific binding of bloodelements to the virus.

These key parameters are in turn driven by important physical andphenotypic characteristics of the specific virus type, which include butare not limited to: the receptor specificity of the virus; the chargecarried on the viral coat; the presence or absence of an envelope; thesize of the viral particle; the immunogenicity of the viral particle;the inflammatory potential of the viral particle; the tumour specificityof the virus; the replicative speed of the virus; and the killingpotency of the virus.

Therefore, the suitability of any given dosing regime will vary withdifferent types of virus and the most appropriate regime may be specificto the type of virus being administered. For example, Zhang et al (2012)describe an Ad5-Ad48 chimeric virus created to reduce hexon binding withblood coagulation factor X in order to eliminate liver sequestration,enhance circulation and decrease toxicity, whilst maintaininganti-tumour activity. Likewise, Shashkova et al (2009) describesignificant differences between wild-type human adenoviral serotypes 5,6, 11, and 35 when investigated as potential anticancer agents. It isthus anticipated that different viral types will behave with significantdifferences when administered systemically to humans and thus theoptimum dosing strategy cannot be predicted a priori without in vivodata and preferably supporting clinical data.

The dose regimen described herein may be particularly suitable toachieve this for group B adenoviruses when compared to, for example thecurrent practice of more regularly spaced and long term repeated dosing.

The objective of an optimised dose regimen for any given oncolyticadenovirus is thus to maximise delivery of the virus to the tumour cellswhilst minimising both the induction of side effects (adverse events)and antiviral immunity, in order to produce a suitable risk benefittreatment profile whilst still allowing for repeat viraladministrations, as appropriate to the therapy. The optimised doseregimen will thus differ between virus types and specifically betweenadenoviral subtypes due to the differences in the viral coat.

Much work has been performed in the prior art based on Ad5, which is asubgroup C adenovirus, the infectivity for which is mediated by theCoxsackie Adenovirus Receptor. When delivered systemically, over 90% ofthe delivered dose is taken up by the liver. The rapid and extensiveloss to the liver reduces the virus uptake by tumours and diminishestherapeutic efficacy. The vast majority of this dose is taken up bycytokine producing innate immune cells such as Kupffer cells, which arespecialised macrophages resident in the liver. Ad5 also exhibits livertoxicity and causes necrosis and subsequent depletion of Kupffer cells.

Shoshkova et al 2009 showed that depletion of Kupffer cells by Ad5increased levels of hepatocyte transduction with subsequent delivery ofAd5 vectors and also suggests that the mechanisms elucidated for Ad5 arenot necessarily relevant to adenoviruses from subgroup B, for exampleAd11 and Ad35. The data therein suggests that subgroup C adenovirusesinteract with Kupffer cells in a similar manner, whereas subgroup Badenoviruses either are not well recognized by Kupffer cells or do notcause death of these cells. In particular, Shoshkova suggests thatpre-dosing with Ad11 based viruses does not have the same beneficialeffect upon Kupffer cells as Ad5. This paper concludes that whilst theremay be some binding for subgroup B adenoviruses (including Ad11) theimpact of this is in fact minimal.

Whilst not wishing to be bound by theory the present inventors believethat, contrary to the prior art suggestions, cytokine producing innateimmune cells such as Kupffer cells may play a role in the clearance ofsubgroup B adenoviruses.

Furthermore, binding of blood coagulation factor X to Ad5 hexon is amechanism of infection of hepatocytes and this mechanism may also berelevant to other adenoviruses in vivo (see for example MolecularTherapy vol. 17 no. 10, 1683-1691 Oct. 2009) but generally is not amechanism of hepatic uptake for adenoviruses from subgroup B.

High global seroprevalence of Ad5 (high Ad5 neutralizing antibody titresin human populations) and certain other adenoviral serotypes represent asignificant concern for the systemic application of high seroprevelanceadeno-based therapies, because such blood-borne viruses can beneutralised by pre-existing antibodies, Vogels et al Journal ofVirology, August 2003 Vol 77, No. 15 page 8263-8271.

Subgroup B adenoviruses have certain inherent advantages in thisrespect, in that they are associated with lower seroprevalence (Stone etal Journal of Virology 2005 Vol 79 No. 8 page 5090-5104) and have lowerinflammatory potential. Initial dosing may thus be far more efficientthan with Ad5, for example. However, the ability to avoid the immunesystem after systemic delivery may still become an issue with repeatdosing. Thus, even with the local suppression of the immune system bythe cancer, avoidance of the immune system is still probably the biggestobstacle to the long term success of oncolytic virus therapy based onsubgroup B adenoviruses. The data generated by the present inventorssupports the position that the therapeutic effect of the oncolyticadenoviruses of subgroup B can thus be improved and/or the eliminationof neutralisation of the adenovirus by the immune system can beminimised by employing an appropriate dosing regimen.

In one embodiment the dosing regimens herein may also minimiseside-effects, for example flu like symptoms and inflammatory responses.

In one embodiment, the replication competent adenovirus is administeredrepeatedly during an early “dosing window” before a specific anti-viralimmune response has been developed, and that later dosing windows mayagain be exploited when the specific anti-viral immune response haswaned once more. That is to say several treatments in a short period oftime followed by a period of time before initiating subsequent treatmentcycles.

Advantageously, by administering the replication competent adenovirus insuch a way, the viral blood levels are sufficient to establish aself-amplifying infection within the tumour (which is known to be animmunosuppressed environment) thereby potentially avoiding the need forchronic repeat administrations with the oncolytic virus. In order toestablish a self-amplifying infection within the tumour, it isbeneficial to maintain the level of virus within the patients' bloodstream at a level above an effective infective concentration for as longas possible but without producing adverse events. This concept is akinto identifying a therapeutic window for the virus, i.e. a range of dosesor dosing regimens where the therapeutic effect is optimised and theside effects are minimised.

This can be achieved by optimising both the dose administered and theinfusion rate of the virus. In one embodiment, the rate of infusion ofthe virus is equal to or greater than the rate of clearance of the virusby the body.

Once an infection is established inside the tumour the virus isrelatively protected from neutralising antibodies and is afforded apotentially permissive environment to replicate and to produce atherapeutic effect without dose limiting toxicities.

In addition, it is hypothesised by the inventors that peaks in virusconcentration (C_(max)) contribute to side effects and that a flatterpharmacological profile may be desirable.

In one embodiment the C_(max) is kept below a specific value, forexample 3×10⁸ DNA copies per ml. It appears that a C_(max) level abovethe relevant threshold is more likely to induce serious adverse eventsor toxicity in some patients.

In one embodiment the rate of infusion has more influence than theabsolute amount of virus administered.

Based on the data generated in the clinic, the present inventors alsobelieve that virus can be delivered at a rate above the rate ofclearance and up to 1.5 to 2×10¹¹ viral particles per minute over aprolonged period such as up to 72 hours or more (wherein the total doseof virus delivered is above 6×10¹² virus particles) without elicitingserious adverse events in the patient.

In one embodiment, the C_(max) of the viral genome in the blood ismaintained at a level of less than 3×10⁸ genomes per ml of blood.

The present inventors have evaluated the initial rate of clearance ofvirus in a number of scenarios and believe that the estimatedα-half-life is in the region of 18 minutes.

The use of prophylactic anti-inflammatories during oncolytic viraltherapy is controversial. On the one hand it has been proposed thattheir use may minimise adverse events and thus enhance tolerability ofoncolytic Newcastle Disease Virus (Lorence et al 2007). On the otherhand, there have been reports that the occurrence of fever may beassociated with enhanced oncolytic efficacy for adenoviruses (Yu et al2007).

The present inventors have found that the use of prophylactic ortherapeutic agents (including anti-inflammatories, steroids,antiemetics, antidiarrheals or analgesics) administered during thistreatment cycle may enhance the tolerability of this regime,particularly allowing for higher or more frequent doses.

In one embodiment, steroids are administered during the treatment cycle.

The present inventors thus hypothesise that six parameters, used eitherindividually or in concert, are important in achieving the goal ofsuitable delivery of an oncolytic subgroup B adenovirus:

-   -   a) the number of virus particles administered with each dose,    -   b) the rate that each virus dose is administered (the number of        viral particles delivered per minute),    -   c) the number of individual doses of virus in the treatment        cycle,    -   d) the interval between each individual dose within the        treatment cycles,    -   e) the use of prophylactic anti-inflammatory medicines during        the treatment cycle, and    -   f) the time period between treatment cycles.

These parameters can be balanced against each other i.e. an increaseddose can be given at a slower infusion rate to off-set the negativeeffect of the increase.

If the dose is too low then the level of viral particles is notsufficient to establish an effective infection of the cancer cells. Ifthe rate of administration is too slow then then the viral particles canreadily be cleared by natural viral sinks (for example cytokineproducing innate immune cells such as hepatic Kupffer cells or bloodcomponents) and an effective infection of the cancer/tumour cells is notachieved. If the viral dose is too high and/or if the rate ofadministration is too fast then the number of adverse events is likelyto increase because of the high concentration of viral particles. Thelatter then induce an inflammatory cytokine response, which may increasethe side-effects experienced by the patient. A moderate infusion ratecan thus optimise the dose delivered.

On average the rate of clearance of type B adenoviruses such as ColoAd1have an α-half-life of about 18 mins.

A single dose of virus may fail to establish an infection, but mayadequately occupy or remove viral sinks (for example cytokine producinginnate immune cells such as hepatic Kupffer cells or blood components).If viral sinks have been adequately occupied or removed, and ifsubsequent doses are administered soon thereafter, the viral kineticsmay be altered for the later doses, with a longer circulating half-lifeand/or higher peak plasma levels. In this case, one or more dosesadministered shortly after the first dose may more effectively establishan effective infection of the cancer cells.

However, if the subsequent doses are administered too far apart (forexample greater than 14 days apart) then the viral sinks may have timeto replenish and the benefit of the prior dose may be lost and/or aspecific anti-viral immune response may have developed. Depletion of,for example cytokine producing innate immune cells such as hepaticKupffer cells with this form of dosing regimen may have an importantsecondary benefit in that Kupffer mediated cytokine release can begreatly reduced on subsequent viral doses such that these doses arebetter tolerated even in the face of higher viral plasma levels.

Thus the present inventors are advocating the administration of a giventreatment cycle over a relatively short period of time, for example asdescribed below.

From the work completed by the present inventors it appears that for agroup B adenovirus multiple doses in a treatment cycle, with each doseadministered over a relatively short period of time, with each doseadministered as a moderately fast infusion, optionally in combinationwith prophylactic agents and with each dose separated by a relativelyshort period of time, is suitable for infecting cancer cells withoncolytic type B adenovirus with minimal toxicity.

Treatment cycles may be repeated as required.

The present inventors have monitored the inflammatory cytokines TNF,gamma interferon, IL-6 and MCP-1 as markers of acute toxicity andbelieve that by the second or subsequent doses, there is reducedtoxicity and increased potential for the virus to infect the cancercells in each case because the non-cancerous viral sinks are eitherremoved or occupied by both the first and the second doses, providedthese doses are administered at an appropriate dose level, rate andfrequency.

In one embodiment three doses are employed in the treatment cycle, andin a further embodiment more than three doses are employed in thetreatment cycle.

In one embodiment, a dose is administered on any or all of days 1, 3, 5,14, and 21.

In another embodiment, a follow-up dose is administered as a maintenanceor booster dose, for example, biweekly, weekly, once every two weeks, orevery 3 weeks, such as every week or every 3 weeks, for a suitableperiod, in particular whilst the treatment is beneficial to the patientas maintenance therapy, for example whilst a patient remains inremission.

The skilled addressee will appreciate that various modifications to thetreatment cycle can be made depending on the needs of the individualpatient.

The present disclosure also extends to a replication capable oncolyticadenovirus of subgroup B for use in treatment of a human patient bysystemically administering at least one dose, such as multiple doses ofa parenteral formulation comprising the adenovirus in a single treatmentcycle, wherein the total dose given in each administration is in therange 1×10¹⁰ to 7×10¹², for example 1×10¹⁰ to 5×10¹² viral particles,and is administered over a period of 1 minute to 90 minutes.

In a further aspect the disclosure extends to use of a replicationcapable oncolytic adenovirus of subgroup B for the manufacture of amedicament for use in the treatment of a human patient by systemicallyadministering at least one dose, such as multiple doses of a parenteralformulation comprising the adenovirus in a single treatment cycle,wherein the total dose given in each dose is in the range 1×10¹⁰ to1×10¹³ for example 1×10¹⁰ to 7×10¹², such as 1×10¹⁰ to 5×10¹², or 6×10¹²viral particles, and is administered over a period of 1 minute to 90minutes.

In one embodiment the first dose in the treatment of a given cycle is alower dose than the dose administered in subsequent treatments in thecycle.

It appears that contrary to Shoshkova's suggestion, based on work inmice, that pre-dosing with Ad11 based viruses does not have a beneficialpriming effect upon cytokine producing innate immune cells such asKupffer cells. In fact, optimisation of the dose and timing betweenadministration of group B oncolytic adenovirus doses may be employed tominimize side effects and hence be beneficial.

In one embodiment the dose administered is 6×10¹², for example over aperiod of 20 to 60 minutes, such as 40 minutes.

In one embodiment a high first and second dose (i.e. which maycorrespond to a normal therapeutic dose) may be desirable to fullyoccupy cytokine producing innate immune cells, such as Kupffer cells(and/or other viral sinks) and thus optimise delivery for subsequentdoses. To put it another way, the first and second dose may be equal.

In one embodiment all the doses administered contain an equal number ofviral particles. This may be particularly advantageous in that itsimplifies manufacture of the viral formulation, reduces the risk ofdosing errors, and may in fact provide a highly effective treatmentregime.

In one embodiment a follow-up cycle of treatment is provided 1 month to6 months after completion of the previous treatment cycle, for example2, 3, 4, 5 months thereafter in order to allow the immune response towane.

In one embodiment a follow-up cycles may be a single dose administeredweekly or bi-weekly, for a period of 1 month to 5 years, such 6, 7, 8,9, 10, 11, 12, 18, 24, 30 or 36 months.

In one embodiment the following-up treatment cycle is initiated withinabout 14 days of administering the last dose in the first treatmentcycle.

The follow-up cycles may also act as maintenance doses, thereby helpingto maintain viral load at a level sufficient to provide a therapeuticeffect.

In one embodiment there are 1, 2, 3, 4, 5 or more subsequent treatmentcycles, for example 1 or 2.

In one embodiment there is only one treatment cycle with no subsequenttreatment cycles.

In one embodiment there is provided a liquid parenteral formulation forinfusion or injection of a replication capable oncolytic subgroup Badenovirus (such as ColoAd1) wherein the formulation provides a dose inthe range of 1×10¹⁰ to 1×10¹⁴ viral particles per volume of dose, suchas 6×10¹² viral particles per dose.

Also disclosed is a method for treating a patient by administering aparenteral formulation according to the present disclosure comprising areplication capable oncolytic subgroup B adenovirus, for examplecontaining a dose described herein, such as 6×10¹² viral particles perdose.

Also disclosed is a method for treating a patient by administeredparenteral formulation according to the present disclosure comprising areplication capable oncolytic subgroup B adenovirus said methodcomprising the co-administration to the patient of one or moresubstances or medicaments selected from the group comprisinganti-inflammatory, steroid, anti-histamines, anti-pyretic medicamentsand fluids for hydration.

Also disclosed is a method of determining when it is suitable toadminister to a subject subsequent cycles of the parenteral formulationaccording to the disclosure comprising a replication capable oncolyticsubgroup B adenovirus, said method comprising the steps of:

-   determining the pre-existing titre of the patient's specific    antiviral immunity prior to a first treatment cycle,    -   serially determining the patient's specific antiviral immunity        subsequent to the first treatment cycle, and    -   delaying any subsequent treatment cycles until the patient's        specific antiviral immunity has reduced to a pre-specified        percentage of baseline.

The term “serially determining” as used herein refers to determining apatient's antiviral immunity at multiple time points, which may beregularly or irregularly spaced apart. The multiple readings obtainedmay be used to generate an average titre over a particular period oftime for example.

The term “pre-specified percentage of baseline” as used herein refers toa viral titre which is defined as a threshold or limit for a particularpatient, taking into account factors such as a baseline measured beforetreatment is initiated, the patient's prognosis, ongoing cancer therapy,any adverse side effects, etc.

In one embodiment, the “pre-specified percentage of baseline” is 90% orless of the patient's baseline viral titre, such as 80% or less, 70% orless, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less,or 10% or less.

In an alternative embodiment, no testing is performed prior toadministering the subsequent treatment cycles.

In one embodiment there is provided use of a glass or plastic syringewith an internal volume in the range of 3 to 50 ml, said syringecontaining a parenteral formulation comprising 1×10¹⁰ to 1×10¹⁴, forexample 1×10¹⁰ to 7×10¹² (such as 1×10¹⁰ to 6×10¹² or 1×10¹⁰ to 5×10¹²,or 1×10¹⁰ to 4×10¹², or 1×10¹⁰ to 3×10¹², or 1×10¹⁰ to 2×10¹², or 1×10¹⁰to 1×10¹²) viral particles, of a replication capable oncolyticadenovirus of subgroup B, wherein the formulation is sterile and wasfilled into the syringe under aseptic conditions, for use in treatment,in particular for use in the manufacture of a medicament which iscapable of injection or intravenous infusion to a human subject.

The skilled person will appreciate that the formulations may include anoverage of the viral particles, for example to compensate for viralparticles that may adhere to the surface of the syringe and which arenot subsequently administered.

Advantageously, such a prefilled syringe would significantly enhance theusability and cost effectiveness for a manufactured oncolytic adenovirusof subgroup B, by removing the need for dose preparation in specialisedpharmacies using expensive resources such as specialised equipment(including extraction hoods) and trained personnel.

The disclosure also extends to pre-filled vials of the said formulation,in particular vials each containing a single dose, in the range definedherein.

In one embodiment the virus formulation is provided in a concentratedform, for example concentrated liquid, suitable for diluting with asterile isotonic diluent, such as saline, glucose or similar locallybefore administration to a patient.

Advantageously, the dosing regimen herein is suitable for delivering atherapeutically effective amount of subgroup B oncolytic virus to thecancer target. In particular the dosing regimen herein may minimiseneutralisation and/or clearance of the oncolytic virus by, for exampleblood born agents, sinks, cytokine producing innate immune cells such asKupffer cells and the immune system. The latter may lead to a betteravailability of the therapeutic dose of the oncolytic virus and,overall, an improved prognosis for the patient and/or improved survival.Advantageously the present regimen may also provide an improved qualityof life for patients by minimising adverse events and/or side effectsduring treatment.

In one embodiment a patient who receives treatment according to thepresent disclosure shows an increased survival rate in comparison to apatient receiving the current standard treatment at the time of filing,for example a statistically significant increase in survival.

In one embodiment a patient who receives treatment according to thepresent disclosure shows a decreased tumour burden, in comparison to thestandard treatment at the time of filing, for example a statisticallysignificant decrease.

In one embodiment the a patient who receives treatment according to thepresent disclosure shows an increased likelihood of going intoremission, in comparison to the standard treatment at the time offiling, for example a statistically significant increase.

In one embodiment the amount or extent of metastasis is reduced, forexample is statistically significantly reduced in a patient who receivestreatment according to the present disclosure in comparison to thestandard treatment at the time of filing.

Whilst not wishing to be bound by theory, it is thought that cells ofthe mononuclear phagocyte system, and in particular cytokine producinginnate immune cells such as Kupffer cells, may be responsible for theclearance of type B oncolytic viruses from the circulation, even thoughthe prior art suggest otherwise.

Furthermore, the mouse studies conducted, by the present inventors,leads them to believe that after the first or second dose in a treatmentregimen, cytokine producing innate immune cells such as the Kupffercells are depleted or occupied such that they are unable to efficientlyclear, for example the third dose and subsequent doses if those dosesare administered in a short time frame after the second dose, oralternatively a lower toxicity may be observed or both. It ishypothesised that the cytokine markers indicate the latter, in that thelevels of the cytokines are not significantly elevated afteradministration of the second or third dose when compared to the firstdose, provided that the doses are administered within a relatively shorttime period. The present inventors take this to be an indication thatthe mechanisms for clearing the virus may be subdued after the first andsecond dose.

Whilst studies in mice do not always parallel what is seen in the humansystem, particularly with viruses, in this instance the humanobservations seem to correlate well with those in the murine modelperformed by the present inventors. The impact of the dosing regimen oncytokine responses and pharmacokinetics of ColoAd1 has also beenexemplified in human subjects, by the present inventors.

As employed herein, “method of treating a patient by systemicallyadministering” is intended to refer to a method of administering atherapeutic agent to a human to effect entry of the entity into thepatient's circulatory system, in particular wherein the treatment isintended to prevent or slow the progression of, ameliorate or cure amalignancy, such as cancer or complications or symptoms associatedtherewith, for example direct administration to the circulatory systemby intravenous administration.

In one embodiment systemic delivery affords the opportunity to treat aprimary tumour, any overt, inaccessible or undiagnosed tumours and/ormetastases. This is particularly advantageous because it may lead to abetter overall prognosis for the patient and/or improved survival.

Thus systemic delivery as employed herein does not refer to treatmentwhich is localised in the tumour or within a body cavity, such as theperitoneal cavity. Examples of systemic delivery include intravenousinfusion and intramuscular and subcutaneous injection.

Parenteral formulation means a formulation designed not to be deliveredthrough the GI tract nor through topical administration. Typicalparenteral delivery routes include injection, implantation or infusion.In one embodiment the formulation is provided in a form for bolusdelivery.

In one embodiment the parenteral formulation is in the form of aninjection. Injection includes intravenous, subcutaneous, intra-tumouralor intramuscular injection. Injection as employed herein means theinsertion of liquid into the body via a syringe. In one embodiment themethod of the present disclosure does not involve intra-tumouralinjection. An injection will generally involve the administration of 150mL of fluid or less over a short period of time, for example 1.5 minutesor less.

In one embodiment the formulation is delivered into the peritonealcavity.

For head and neck cancer, or brain metastases of epithelial cancers,intracranial injection may be necessary.

In one embodiment the parenteral formulation is in the form of aninfusion.

Infusion as employed herein means the administration of fluids at aslower rate by drip, infusion pump, syringe driver or equivalent device.In one embodiment the infusion is administered over a period in therange of 1.5 minutes to 90 minutes, such as 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes.

In one embodiment the volume of formulation administered is 100 mLs orless, in particular 50 mLs or less, for example 30 mls, 10 ml, 5 ml orless, such as 3 ml, such as administered by a syringe driver. The lattermay be referred to as a slow injection.

In one embodiment the infusion is delivered at a rate in the range of0.5 to 6 ml per minute, for example 0.75 ml per minute. In oneembodiment, the infusion is delivered at a rate in the range of 2×10⁹ to2×10¹² virus particles (VP) per minute, for example 1.5×10¹¹ VP perminute.

In one embodiment the injection is administered as a slow injection, forexample over a period of 1.5 to 30 or 1.5 to 40 minutes.

In one embodiment the formulation is for intravenous administration.This route is particularly effective for delivery of oncolytic virusbecause it allows rapid access to the majority of the organs and tissueand is particular useful for the treatment of metastases, for exampleestablished metastases especially those located in highly vascularisedregions such as the liver and lungs.

In one embodiment a combination of administration methods are employed,for example IV and intra-tumourally or intraperitoneally andintra-tumourally, or IV and intra-peritoneally.

Thus in one embodiment systemic administration of the present disclosuremay be employed in combination with other routes of administration, suchas intra-tumoural administration either concomitantly or sequentially,for example a first pre-treatment cycle may be intra-tumoural and thesecond treatment cycle may be systemic according to the presentdisclosure. Alternatively, the first treatment cycle may be according tothe present disclosure and subsequent cycles or boosts may beintra-tumoural, as appropriate. Therapeutic formulations typically willbe sterile and stable under the conditions of manufacture and storage.The composition can be formulated as a solution, microemulsion,liposome, or other parenteral formulation suitable for administration toa human and may be formulated as a pre-filled device such as a syringeor vial, particular as a single dose.

In one embodiment 2 or more doses are employed in the treatment cycle,for example 2, 3, 4, 5 or 6 doses are employed in each treatment cycleand for example may be provided as a kit.

Each dose administered in a given treatment cycle may be referred toherein as a treatment.

In one embodiment a lower first dose is employed in comparison to thesubsequent doses administered in the cycle, for example a lower dose maybe in the range of 30-95% of the subsequent dose or doses, for example50, 60, 70 or 80%.

In one embodiment a higher first is employed in comparison to thesubsequent doses administered in the cycle, which may be desirable tofull occupy cytokine producing innate immune cells such as the Kupffercells and thus optimise delivery for subsequent doses.

A higher dose means more than 100% of the subsequent dose, for example105 to 150% of the subsequent dose, such as 110%, 115%, 120%, 125%,130%, 135%, 140% or 145% of the subsequent dose.

In one embodiment 1, 2, 3 or all the doses administered contains anequal number of viral particles. This may be particularly advantageousin that it simplifies manufacture of the viral formulation and may infact provide a highly effective treatment regime.

In one embodiment the “same dose” i.e. the same number of viralparticles are administered in one or more doses, such as all the dosesin a treatment cycle, however, the doses may be administered atdifferent rates, for example as described herein.

Treatment cycle as employed herein is the period of treatment between aperiod of rest in a course of treatment repeated in accordance with aschedule with periods of rest there-between.

A treatment cycle generally refers to multiple (i.e. at least two)treatments administered as part of a program or schedule of treatment,administered over a relatively short period of time, for example about 1to 4 weeks, such as 3 weeks, 2 weeks, or 1 week. Generally, a giventreatment cycle will be a part of a larger treatment regime.

In one embodiment the treatment cycle is a period of 14 days or less,for example 10, 9, 8, 7 or 5 days, such as 7 or 5 days.

In one embodiment each further dose or doses is/are administered atapproximately 48 hour intervals, such as every 40 to 56 hours. This isadvantageous since it allows dosing to occur within a normal workingweek or within an outpatient setting.

In one embodiment the first dose is administered on day 1 and thefurther therapeutic doses are administered every second day thereafter,such as on days 1, 3, 5, 7, 9, 11 and 13, or once every approximately 48hours thereafter, such as every 40 to 56 hours.

In one embodiment the plasma levels of virus in the patient afteradministration of the dose (such as the second or subsequent dose) is atleast 2×10⁶ viral particles per ml, for example for a period of 15minutes or longer, for example 20, 30, 40, 50, 60 minutes or more.

In vitro studies performed by the inventors (see FIG. 1) suggest thatfor virus particles in whole human blood at 37° C., killing drops below50% at <2×10⁶ particles ml. Furthermore, the inventors have been able toshow the presence of live viral particles in patient blood using plaqueassays when viral genome levels are above for example 1.6e6 to 1e8, andcan be consistently detected. In one embodiment there is at least 14days between treatment cycles.

The formulation will generally comprise a pharmaceutically acceptablediluent or carrier, for example a non-toxic, isotonic carrier that iscompatible with the virus, and in which the virus is stable for therequisite period of time.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a dispersant or surfactant such as lecithin or a non-ionicsurfactant such as polysorbate 80 or 40.

In dispersions the maintenance of the required particle size may beassisted by the presence of a surfactant. Examples isotonic agentsinclude sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride in the composition.

In one embodiment a sterile isotonic diluent such as saline or glucose(for example 5% glucose is employed).

In one embodiment parenteral formulations employed in the method maycomprise one or more of the following a buffer, for example4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, a phosphate buffer,and/or a Tris buffer, a sugar for example dextrose, mannose, sucrose orsimilar, a salt such as sodium chloride, magnesium chloride or potassiumchloride, a detergent such as a non-ionic surfactant such as Briji®,PS-80, PS-40 or similar. The formulation may also comprise apreservative such as EDTA or ethanol or a combination of EDTA andethanol, which are thought to prevent one or more pathways of possibledegradation.

In one embodiment the formulation will comprise purified oncolyticvirus, for example 1×10¹⁰ to 1×10¹⁴ viral particles per dose, such as1×10¹⁰ to 7×10¹² viral particles per dose, in particular 1×10¹⁰ to1×10¹² viral particles per dose, including overage as necessary.

In one embodiment the formulation according to the present disclosurecomprises 6×10¹² viral particles.

In one embodiment the concentration of virus in the formulation is inthe range 2×10⁸ to 2×10¹⁴ vp/mL, such as 2×10¹² vp/ml.

In one embodiment the parenteral formulation comprises glycerol.

In one embodiment the formulation comprises oncolytic adenovirus fromsubgroup B, HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid),glycerol and buffer.

In one embodiment the parenteral formulation consists of virus, HEPESfor example 5 mM, glycerol for example 5-20% (v/v), hydrochloric acid,for example to adjust the pH into the range 7-8 and water for injection.

In one embodiment 0.7 mL of ColoAd1 at a concentration of 2×10¹² vp/mLis formulated in 5 mM HEPES, 20% glycerol with a final pH of 7.8.

Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, monostearate salts and gelatin.

Thus the oncolytic adenoviruses employed herein may be administered in atime release formulation, for example in a composition which includes aslow release polymer. The oncolytic adenovirus can be prepared withcarriers that will protect it against neutralisation and/or preventrapid release, such as a controlled release formulation, such asimplants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers(PLG). Biocompatible non-degradable polymers such as Polyethylene glycoland poly(N-(2-hydroxypropyl)methacrylamide) can also be used. Manymethods for the preparation of such formulations are known to thoseskilled in the art.

Sterile injectable solutions can be prepared by incorporating theoncolytic adenovirus in the required amount in an appropriate solvent,for example with one or a combination of ingredients described herein,as relevant, followed by filtered sterilisation. Generally, dispersionsare prepared by incorporating the oncolytic adenovirus into a sterilevehicle which contains a basic dispersion medium and the required otheringredients.

Generally the parenteral formulation according to the disclosure is asterile liquid formulation, such as an aqueous formulation,substantially free of particulates, for example prepared aseptically andsterilised by passing through a 0.2 micron filter.

In one embodiment the therapeutic parenteral formulation is administeredto minimise the contact of the formulation with the epidermis of thepatient, for example employing a sheathed needle or via a cannula. Thisprecaution is thought to minimise the immune response of the patient tothe oncolytic virus, for example by minimising contact with Langerhancells in the skin. Replication capable as employed herein is a virusthat can replicate in a host cell. In one embodiment replication capableencompasses replication competent and replication selective viruses.

Replication competent as employed herein is intended to mean anoncolytic adenovirus that is capable of replicating in a human cell,such as a cancer cell, without any additional complementation to thatrequired by wild-type viruses, for example without relying on defectivecellular machinery. That is, they are tumour selective by infectingtumour cells in preference to non-tumour cells. ColoAd1 is an example ofa replication competent virus.

Replication selective or selective replication as employed herein isintended to mean an oncolytic adenovirus that is able to replicate incancer cells employing an element which is specific to said cancer cellsor upregulated therein, for example defective cellular machinery, suchas a p53 mutation, thereby allowing a degree of selectivity overhealthy/normal cells.

Oncolytic subgroup B adenovirus as employed herein refers to anadenovirus comprising at least the hexon and fiber from subgroup B (seeShenk et al and Table 1) that preferentially infects and/or lyses tumourcells compared with normal cells. Thus an oncolytic subgroup Badenovirus as employed herein includes a chimeric, a mutant or avariant, with the fiber and hexon of a group B adenovirus and whichretains oncolytic properties.

Adenovirus or adenoviral serotype as used herein refers to any of thehuman adenoviral serotypes currently known (51) or isolated in thefuture. See for example, Strauss (1984) and Shenk (2001). Adenovirusserotypes are classified into subgroups as shown in Table 1.

Table 1 shows the division of adenovirus serotvnes:

Subgroup Adenoviral Serotype A 12, 18, 31 B 3, 7, 11, 14, 16, 21, 34,35, 51 C 1, 2, 5, 6 D 8-10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39,42-50 E 4 F 40, 41

Examples of subgroup B viruses include Ad11 (wild-type) such as Ad11aand Ad11p (Genbank Accession No: AF532578) and the chimeric adenovirusColoAd1. The latter is disclosed in WO 2005/118825 and the full sequencefor the virus is provided in SEQ ID NO: 1 therein.

Thus in one embodiment the virus employed in the method according to thepresent disclosure is a chimeric virus.

Chimeric adenovirus as employed herein refers to adenoviruses which haveDNA from two or more different adenovirus serotypes such as thosegenerated using the method of WO2005/118825 which is incorporated hereinby reference.

In one embodiment the chimeric adenovirus is ColoAd1. ColoAd1 is thoughtto kill tumour cells by a mechanism which more closely resemblesnecrosis than apoptosis (unpublished data produced at the University ofOxford). This has a number of potential beneficial effects (Kirn et al2001; Small et al 2006; Reid et al 2002; Liu et al 2007; Ferguson et al2012):

-   -   ColoAd1 has been shown to be potent in multi-drug resistant        cancer cell lines and in cancer stem-cell like cells, which are        known to have a resistance to apoptosis;    -   An inflammatory necrotic cell death may be more suitable for the        generation of a specific anti-tumoural immune response;    -   ColoAd1 exits tumour cells very rapidly, even before target cell        death, and may thus have enhanced ability to spread.

ColoAd1 is a chimera of Ad11 and Ad3 but has an outer capsule which isentirely homologous with that of Ad11. The viral kinetics, inflammatorypotential and immunological characteristics of ColoAd1 thus most closelyresemble and predict those of Ad11 and other subgroup B adenoviruses.

In one embodiment the oncolytic virus employed in the method of thepresent disclosure is deleted in the E3 and/or E4 region or partthereof. This may be beneficial because it may allow more rapidreplication of the virus in vivo.

In addition the E3 deletion may contribute to the rapid clearance of thevirus from non-cancer cells as the E3 region encoded proteins which maybe relevant to avoiding the immunity of the host.

In one embodiment the virus employed in the method of the presentdisclosure is based on Ad11 or derived therefrom such that the hexon andfibre are substantially similar to Ad11, such as Ad11p. Furthermoresince the serotype designation of adenovirus is based on the exteriorproperties of the virus i.e. hexon and fibre properties, the presentdisclosure is useful in type B adenovirus which have similar surfaceproperties.

In one embodiment the type B adenovirus is OvAd1 or OvAd2 which aredisclosed in SEQ ID NO: 1 and SEQ ID NO: 2 respectively inWO2008/080003, incorporated herein by reference.

Substantially similar as employed herein refers to an amino acidsequence for a relevant protein or proteins which is/are at least 95%identical (e.g. 96, 97, 98, 99 or 100% identical) over the “whole” ofthe particular protein. The protein(s) being compared may be part of alarger entity but the comparison will be the whole length of therelevant fragment or component.

Adenovirus type 5 (Ad5) generally enter the cell via thecoxsackie-adenovirus receptor (CAR). However, Adenovirus serotype 11(Ad11) is a subgroup B adenovirus that targets a different receptor(CD46) which is expressed at low levels in all nucleated cells. Innormal cells CD46 is often hidden on the basolateral surfaces of cellsand is thus not available for virus binding (Varela J C, et al IntlCancer 2008 Sep. 15; 123(6):1357-63; Maisner et al., 1997). However, intumour cells it typically has enhanced surface expression, particularlyin more advanced and aggressive tumours (Kinugasa et al., 1999).Therefore, Ad11 efficiently infects carcinoma cell lines, for examplefrom lung epithelial carcinoma (A549 cells), hepatoma (HepG2), prostaticcancer (DU 145 and LNCaP), laryngeal cancer (Hep2) and breast cancer(CAMA and MG7) and also to glioblastoma, medulloblastoma andneuroblastoma cells (Mei et al 2003). Thus Ad11 preferentially infectstumour cells and viruses derived therefrom are thought to be useful inthe treatment of at least one or more of the above cancers. As a chimeraof Ad11 and Ad3, ColoAd1 shares these characteristics with Ad11.

In one embodiment a virus employed in the method of the presentdisclosure comprises a transgene (in particular one or more transgenes),for example a therapeutic transgene, for expression in vivo. A transgenegene as employed herein is intended to refer to a gene not found in theparent or wild type virus. Such genes may perform a function as a markeror reporter for tracking efficacy of viral infection. Alternatively thegene may perform a role in improving the efficacy of the virus.Alternatively the gene may deliver a cytotoxic agent to the cell.

The therapeutic transgene may express a therapeutic agent in the cell,for example siRNA; shRNA; a polypeptide; tumour associated antigen(TAA), cytokine; antibody; or an anti-angiogenesis factor.

Examples of therapeutic antibodies include anti-VGEF antibodies such asbevacizumab, anti-EGFR antibodies such as cetuximab, an anti-CD20antibody such as rituximab, or an immune system activator modulator suchas anti-CTLA4 (e.g. ipilimumab), anti-PD-1 and anti-PD-L1 amongstothers. Single chain antibodies, antibody subunits, antibody fragmentsand TRAPs may also be encoded as well as full length antibodies.Importantly for the current disclosure, the inclusion of these proteinsdoes not change the surface properties of the virus and therefore canreadily be incorporated into the genome without deleterious effects uponthe dosing as described herein whilst providing additional therapeuticmechanisms for attacking the cancer cells.

Examples of cytokines include interferon-alpha, interferon-gamma andIL-2 amongst others.

As the RNA, antibody, polypeptide, TAA or cytokine will be expressed inthe tumour it is thought that this presents an opportunity to change themicroenvironment of the tumour but avoid systemic side effects of thedelivered agent. For example, it may be possible to stimulate the localimmune system to attack the cancer. It is possible to modulate thislocal effect by altering whether or not the RNA, antibody, polypeptide,TAA or cytokine is secreted from the cell and when during the viral lifecycle it is expressed.

In one embodiment the transgene encodes thymidine kinase, for examplefrom a non-human origin or cytosine deaminase, for example frombacterial origin or from a yeast.

In one embodiment the antibody, polypeptide or cytokine or similar isnon-human in origin and is not humanised. The latter is not likely todetrimentally effect the activity of the entity in the cancer cell andhas the advantage that material that may escape the cancer cell willattract the attention of the immune system locally and will be rapidlycleared.

In one embodiment the virus encodes and expresses in vivo a visible orvisualisable protein, for example a fluorescent protein, such as GFP orsimilar. Given the virus selectively infects cancerous cells, when itexpresses a visible or visualisable protein then it can be used tohighlight the area of cancerous tissue for resection or radiation.

In one embodiment, the viruses may be armed with therapeutic genescapable of eliciting anti-tumour immune function, inhibition of tumourneovascularization, or prodrug activation. Therapeutic dose as employedherein refers to the amount of oncolytic adenovirus that is suitable forachieving the intended therapeutic effect when employed in a suitabletreatment regimen, for example ameliorates symptoms or conditions of adisease. A dose may be considered a therapeutic dose in the treatment ofcancer or metastases when the number of viral particles may besufficient to result in the following: tumour or metastatic growth isslowed or stopped, or the tumour or metastasis is found to shrink insize, and/or the life span of the patient is extended. Suitabletherapeutic doses are generally a balance between therapeutic effect andtolerable toxicity, for example where the side-effect and toxicity aretolerable given the benefit achieved by the therapy.

In one embodiment the therapeutic dose range does not have a doselimiting toxicity.

Dose limiting toxicity as employed herein means the appearance of sideeffects during treatment that are severe enough to prevent any one ofthe following: further increase in dosage, frequency or strength or toprevent continuation of treatment at any dosage level. Toxicity effectswhich are intolerable, for example associated with a high dose mean thelatter is not suitable for use as a therapeutic dose in the context ofthe present disclosure.

In one embodiment pre-existing immunity to the Ad11 capsid is weakpermitting effective administration of further therapeutic doses on orafter day 7.

In one embodiment the poor immune stimulatory properties of the Ad11capsid permits effective administration of further therapeutic doses onor after day 7.

In one embodiment intravenous delivery of the virus is less immunogenicin terms of antiviral immunogenicity than sub-cutaneous or intramusculardelivery of virus.

It is generally believed that the toxicity of Ad11 may be lower thancertain other adenoviruses, such as Ad5. This together with the lowerseroprevalence is beneficial but this may not be sufficient to allowAd11 to evade immune responses. Even though the literature suggests thatthe subgroup B adenoviruses are not toxic to liver cells it may be thatmacrophages in the lungs, liver (Kupffer cells) and spleen clearoncolytic viruses after systemic delivery.

It is thought that the rapid delivery of at least two doses of theoncolytic virus may be beneficial in generating sufficient levels ofvirus that are sustained for a period which allows adequate infection ofthe target cells, namely cancer cells.

Providing at least two doses in quick succession may allow one or morethe following beneficial events to occur a) the immune mechanisms areoccupied by the first dose, which may then allow the second dose toescape the full onslaught of the immune system to reach the targetand/or b) at least two doses in quick succession allow thebiodistribution of the virus to reach sufficient levels for a sufficientperiod to reach the target cells in vivo, either way once the virusreaches and infects the target cells it is able to replicate.

Biodistribution as employed herein means the distribution in vivo.

Whilst not wishing to be bound by theory the inventors believe that thefirst dose of virus may down regulate clearance, for example mechanismssuch as those employing cytokine producing innate immune cells such asKupffer cells thereby improving the bioavailablity for the furthertherapeutic dose(s). The first dose of virus may thus “deplete” thephagocytic ‘sinks’ for circulating virus thereby achieving betterdelivery and/or increased efficacy. Depleting the phagocytic sinks alsoreduces the tendency to release cytokines on subsequent doses and thusallows higher viral blood levels to be achieved without excessivetoxicity.

Bioavailability as employed herein means the amount of virus availableto perform its intended therapeutic function in vivo.

In one embodiment the method herein wherein at least three doses areadministered minimises side-effects and/or toxicity in the patient.

In one embodiment the adenovirus is stealthed by coating said virus witha polymer, for example to at least partially avoid the patient's immunesystem.

Stealthed as employed herein means that the adenovirus's exteriorsurface has been modified to avoid the patient's immune response, forexample employing a polymer. Examples of suitable polymers are disclosedin WO98/19710, WO00/74722, WO2010/067041, WO2010/067081, andWO2006/008513 incorporated herein by reference.

In one embodiment the oncolytic virus is conjugated to a cytotoxic orimmunomodulatory agent.

In one embodiment the oncolytic adenovirus is provided which ispegylated, for example to reduce immunogenenicity and/or increasehalf-life.

In one embodiment the method of treatment is for use in the treatment ofa tumour.

Tumour as employed herein is intended to refer to an abnormal mass oftissue that results from excessive cell division that is uncontrolledand progressive, also called a neoplasm. They may be either benign (notcancerous) or malignant. Tumour encompasses all forms of cancer andmetastases.

In one embodiment the tumour is a solid tumour. The solid tumour may belocalised or metastasised.

In one embodiment the tumour is of epithelial origin.

In one embodiment the tumour is a solid tumour.

In one embodiment the tumour is a malignancy, such as colorectal cancer,hepatoma (liver cancer), prostate cancer, pancreatic cancer, breastcancer, ovarian cancer, thyroid cancer, renal cancer, bladder cancer,head and neck cancer or lung cancer.

In one embodiment the tumour is a colorectal malignancy.

Malignancy as employed herein means cancerous cells.

In one embodiment the cancer is colorectal cancer and/or metastaticforms thereof such as liver metastasis.

In one embodiment the cancer is liver cancer and/or metastatic formsthereof.

In one embodiment the cancer is lung cancer and/or metastatic formsthereof.

In one embodiment the cancer is ovarian cancer and/or metastatic formsthereof, such as lung metastasis.

In one embodiment the cancer is renal cancer and/or metastatic formsthereof.

In one embodiment the cancer is bladder cancer and/or metastatic formsthereof.

In one embodiment the cancer is throat cancer.

In one embodiment the cancer is skin cancer, such as melanoma. In oneembodiment the cancer is Leukemia. In one embodiment the cancer isglioblastoma, medulloblastoma or neuroblastoma. In one embodiment thecancer is a neuroendocrine cancer. In one embodiment the cancer isHodgkin's or non-Hodgkins lymphoma.

In one embodiment the oncolytic adenovirus is employed in the treatmentor prevention of metastasis.

In one embodiment the oncolytic adenoviruses described herein aresuitable for the treatment of cancerous cells that have migrated to thelymph node. The present inventors have shown that oncolytic virusadministered to colorectal cancer patients can infect cancerous cellsthat have migrated to the lymph nodes.

In one embodiment the virus, formulations and regimens according to thepresent disclosure are suitable for treating abnormal pre-cancerouscells.

In one embodiment the method or formulation herein is employed in thetreatment of drug resistant cancers.

In one embodiment the method or formulation is employed in to sensitisedrug resistant to cancers to said drugs.

Cancer Types in More Detail

Lung Cancer

Lung cancers are classified according to histological type and arecategorized by the size and appearance of the malignant cells seen by ahistopathologist under a microscope. For therapeutic purpose, two broadclasses are distinguished: non-small cell lung carcinoma and small celllung carcinoma.

In one embodiment the epithelial cancer is lung cancer, for examplesmall-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC).

Non-small-cell lung carcinoma—The three main subtypes of NSCLC areadenocarcinoma, squamous-cell carcinoma and large-cell carcinoma.

Nearly 40% of lung cancers are adenocarcinoma, which usually originatesin peripheral lung tissue. A subtype of adenocarcinoma, thebronchioloalveolar carcinoma, is more common in female never-smokers,and may have a better long term survival.

Squamous-cell carcinoma accounts for about 30% of lung cancers. Theytypically occur close to large airways. A hollow cavity and associatedcell death are commonly found at the center of the tumour. About 9% oflung cancers are large-cell carcinoma. These are so named because thecancer cells are large, with excess cytoplasm, large nuclei andconspicuous nucleoli.

Small-cell lung carcinoma—In small-cell lung carcinoma (SCLC), the cellscontain dense neurosecretory granules (vesicles containingneuroendocrine hormones), which give this tumour anendocrine/paraneoplastic syndrome association. Most cases arise in thelarger airways (primary and secondary bronchi). These cancers growquickly and spread early in the course of the disease. Sixty to seventypercent have metastatic disease at presentation.

In one embodiment the cancer is non-small lung carcinoma.

Liver Cancer

In one embodiment the cancer is liver cancer, for example a livermetastasis from a primary cancer, for example colon cancer, which hasspread to the liver. In one embodiment the liver cancer ishepatocellular carcinoma (HCC).

Renal Cancer

In one embodiment there is provided treatment of renal cancer, forexample renal cell carcinoma and/or urothelial cell carcinoma using anoncolytic adenovirus as disclosed herein. Other examples of renal cancerinclude squamous cell carcinoma, juxtaglomerular cell tumour (reninoma),angiomyolipoma, renal oncocytoma, Bellini duct carcinoma, clear-cellsarcoma of the kidney, mesoblastic nephroma, Wilms' tumour, mixedepithelial stromal tumour, clear cell adenocarcinoma, transitional cellcarcinoma, inverted papilloma, renal lymphoma, teratoma, carcinosarcoma,and carcinoid tumour of the renal pelvis.

Bladder Cancer

In one embodiment the cancer is bladder cancer, for example is any ofseveral types of malignancy arising from the epithelial lining (i.e.,the urothelium) of the urinary bladder. About 90% of bladder cancers aretransitional cell carcinoma. The other 10% are squamous cell carcinoma,adenocarcinoma, sarcoma, small cell carcinoma, and secondary depositsfrom cancers elsewhere in the body. The staging of is given below.

T (Primary Tumour)

-   -   TX Primary tumour cannot be assessed    -   T0 No evidence of primary tumour    -   Ta Non-invasive papillary carcinoma    -   Tis Carcinoma in situ (‘flat tumour’)    -   T1 Tumour invades subepithelial connective tissue    -   T2a Tumour invades superficial muscle (inner half)    -   T2b Tumour invades deep muscle (outer half)    -   T3 Tumour invades perivesical tissue:    -   T3a Microscopically    -   T3b Macroscopically (extravesical mass)    -   T4a Tumour invades prostate, uterus or vagina    -   T4b Tumour invades pelvic wall or abdominal wall

N (Lymph Nodes)

-   -   NX Regional lymph nodes cannot be assessed    -   N0 No regional lymph node metastasis    -   N1 Metastasis in a single lymph node 2 cm or less in greatest        dimension    -   N2 Metastasis in a single lymph node more than 2 cm but not more        than 5 cm in greatest dimension, or multiple lymph nodes, none        more than 5 cm in greatest dimension    -   N3 Metastasis in a lymph node more than 5 cm in greatest        dimension

M (Distant Metastasis)

-   -   MX Distant metastasis cannot be assessed    -   M0 No distant metastasis    -   M1 Distant metastasis.

The current disclosure extends to any stage of bladder cancer.

Ovarian Cancer

In an independent aspect the present disclosure relates to ColoAd1, aformulation of the same or a combination therapy comprising ColoAd1, foruse in treating ovarian cancer, for example administering atherapeutically effective amount of ColoAd1 to a patient with ovariancancer, for example employing a dosing regimen described herein.

There are more than 30 different types of ovarian cancer which areclassified according to the type of cell from which they start.Cancerous ovarian tumours can start from three common cell types:

-   -   Surface Epithelium—cells covering the lining of the ovaries    -   Germ Cells—cells that are destined to form eggs    -   Stromal Cells—Cells that release hormones and connect the        different structures of the ovaries

The present disclosure relates to treatment of ovarian cancer from anysource, for example as described herein, in particular epithelium cells.Epithelial ovarian carcinomas (EOCs) account for 85 to 90 percent of allcancers of the ovaries.

Common Epithelial Tumours—Epithelial ovarian tumours develop from thecells that cover the outer surface of the ovary. Most epithelial ovariantumours are benign (noncancerous). There are several types of benignepithelial tumours, including serous adenomas, mucinous adenomas, andBrenner tumours. Cancerous epithelial tumours are carcinomas—meaningthey begin in the tissue that lines the ovaries. These are the mostcommon and most dangerous of all types of ovarian cancers.Unfortunately, almost 70 percent of women with the common epithelialovarian cancer are not diagnosed until the disease is advanced in stage.

There are some ovarian epithelial tumours whose appearance under themicroscope does not clearly identify them as cancerous. These are calledborderline tumours or tumours of low malignant potential (LMP tumours).The method of the present disclosure includes treatment of the latter.

Germ Cell Tumours—Ovarian germ cell tumours develop from the cells thatproduce the ova or eggs. Most germ cell tumours are benign(non-cancerous), although some are cancerous and may be lifethreatening. The most common germ cell malignancies are maturingteratomas, dysgerminomas, and endodermal sinus tumours. Germ cellmalignancies occur most often in teenagers and women in their twenties.Today, 90 percent of patients with ovarian germ cell malignancies can becured and their fertility preserved.

Stromal Tumours—Ovarian stromal tumours are a rare class of tumours thatdevelop from connective tissue cells that hold the ovary together andthose that produce the female hormones, estrogen and progesterone. Themost common types are granulosa-theca tumours and Sertoli-Leydig celltumours. These tumours are quite rare and are usually consideredlow-grade cancers, with approximately 70 percent presenting as Stage Idisease (cancer is limited to one or both ovaries).

Primary Peritoneal Carcinoma—The removal of one's ovaries eliminates therisk for ovarian cancer, but not the risk for a less common cancercalled Primary Peritoneal Carcinoma. Primary Peritoneal Carcinoma isclosely rated to epithelial ovarian cancer (most common type). Itdevelops in cells from the peritoneum (abdominal lining) and looks thesame under a microscope. It is similar in symptoms, spread andtreatment.

Stages of Ovarian Cancer

Once diagnosed with ovarian cancer, the stage of a tumour can bedetermined during surgery, when the doctor can tell if the cancer hasspread outside the ovaries. There are four stages of ovariancancer—Stage I (early disease) to Stage IV (advanced disease). Thetreatment plan and prognosis (the probable course and outcome of yourdisease) will be determined by the stage of cancer you have.

Following is a description of the various stages of ovarian cancer:

-   Stage I—Growth of the cancer is limited to the ovary or ovaries.-   Stage IA—Growth is limited to one ovary and the tumour is confined    to the inside of the ovary. There is no cancer on the outer surface    of the ovary. There are no ascites present containing malignant    cells. The capsule is intact.-   Stage IB—Growth is limited to both ovaries without any tumour on    their outer surfaces. There are no ascites present containing    malignant cells. The capsule is intact.-   Stage IC—The tumour is classified as either Stage IA or IB and one    or more of the following are present: (1) tumour is present on the    outer surface of one or both ovaries; (2) the capsule has ruptured;    and (3) there are ascites containing malignant cells or with    positive peritoneal washings.-   Stage II—Growth of the cancer involves one or both ovaries with    pelvic extension.-   Stage IIA—The cancer has extended to and/or involves the uterus or    the fallopian tubes, or both.-   Stage IIB—The cancer has extended to other pelvic organs.-   Stage IIC—The tumour is classified as either Stage IIA or IIB and    one or more of the following are present: (1) tumour is present on    the outer surface of one or both ovaries; (2) the capsule has    ruptured; and (3) there are ascites containing malignant cells or    with positive peritoneal washings.-   Stage III—Growth of the cancer involves one or both ovaries, and one    or both of the following are present: (1) the cancer has spread    beyond the pelvis to the lining of the abdomen; and (2) the cancer    has spread to lymph nodes. The tumour is limited to the true pelvis    but with histologically proven malignant extension to the small    bowel or omentum.-   Stage IIIA—During the staging operation, the practitioner can see    cancer involving one or both of the ovaries, but no cancer is    grossly visible in the abdomen and it has not spread to lymph nodes.    However, when biopsies are checked under a microscope, very small    deposits of cancer are found in the abdominal peritoneal surfaces.-   Stage IIIB—The tumour is in one or both ovaries, and deposits of    cancer are present in the abdomen that are large enough for the    surgeon to see but not exceeding 2 cm in diameter. The cancer has    not spread to the lymph nodes.-   Stage IIIC—The tumour is in one or both ovaries, and one or both of    the following is present: (1) the cancer has spread to lymph nodes;    and/or (2) the deposits of cancer exceed 2 cm in diameter and are    found in the abdomen.-   Stage IV—This is the most advanced stage of ovarian cancer. Growth    of the cancer involves one or both ovaries and distant metastases    (spread of the cancer to organs located outside of the peritoneal    cavity) have occurred. Finding ovarian cancer cells in pleural fluid    (from the cavity which surrounds the lungs) is also evidence of    stage IV disease.

In one embodiment the ovarian cancer is: type I, for example IA, IB orIC; type II, for example IIA, IIB or IIC; type III, for example IIIA,IIIB or IIIC; or type IV.

The present disclosure relates to treatment of any stage of ovariancancer, in particular as described herein.

Combination Therapy

In one embodiment the virus is administered in combination with theadministration of a further cancer treatment or therapy.

“In combination” as employed herein is intended to encompass where theoncolytic virus is administered before, concurrently and/or post cancertreatment or therapy.

In one embodiment the oncolytic adenovirus is employed in combinationwith high intensity focused ultrasound (HIFU) treatment.

Cancer therapy includes surgery, radiation therapy, targeted therapyand/or chemotherapy.

Cancer treatment as employed herein refers to treatment with atherapeutic compound or biological agent, for example an antibodyintended to treat the cancer and/or maintenance therapy thereof.

In one embodiment the cancer treatment is selected from any otheranti-cancer therapy including a chemotherapeutic agent, a targetedanticancer agent, radiotherapy, radio-isotope therapy or any combinationthereof.

In a further independent aspect the present disclosure relates to acombination therapy comprising oncolytic type B adenovirus, such asColoAd1, and a chemotherapeutic agent which does not interfere with theadenovirus activity. Type B adenovirus, such as ColoAd1 as employedherein includes formulations thereof, for example pharmaceuticalformulations thereof.

Activity as employed herein refers to any beneficial property orcharacteristic of the virus, for example the oncolytic activity and orthe ability of the virus to replicate in cancer cells, such as viralreplication in vivo.

In one embodiment the ColoAd1 in the combination therapy is dosedaccording to a regimen described herein.

Generally, the combination therapy will be provided as a formulation ofthe adenovirus and a formulation of the chemotherapeutic agent. Thus theadministration of the adenovirus and the chemotherapeutic will suitablybe separate events. These administrations may be on the same ordifferent days.

In one embodiment the adenovirus is administered in a suitable regimeone week and the chemotherapeutic is administer a following week, forexample the next.

In one or more embodiments the chemotherapeutic agent and the adenovirusmay have a synergistic therapeutic effect.

The oncolytic adenovirus may be used as a pre-treatment to the therapy,such as a surgery (neoadjuvant therapy), to shrink the tumour, to treatmetastasis and/or prevent metastasis or further metastasis. Theoncolytic adenovirus may be used after the therapy, such as a surgery(adjuvant therapy), to treat metastasis and/or prevent metastasis orfurther metastasis.

Concurrently as employed herein is the administration of the additionalcancer treatment at the same time or approximately the same time as theoncolytic adenovirus formulation. The treatment may be contained withinthe same formulation or administered as a separate formulation.

In one embodiment the virus is administered in combination with theadministration of a chemotherapeutic agent, for example as describedherein, such as paclitaxel, abraxane or similar.

Chemotherapeutic agent as employed herein is intended to refer tospecific antineoplastic chemical agents or drugs that are selectivelydestructive to malignant cells and tissues. For example alkylatingagents, antimetabolites, anthracyclines, plant alkaloids, topoisomeraseinhibitors, and other antitumour agents. Other examples of chemotherapyinclude doxorubicin, 5-fluorouracil (5-FU), paclitaxel, capecitabine,irinotecan, and platins such as cisplatin and oxaliplatin. The preferreddose may be chosen by the practitioner based on the nature of the cancerbeing treated.

Surprisingly the present inventors have established that certain classesof therapeutic agents can inhibit viral replication, for exampletopoisomerase or parp inhibitors, may inhibit the replication of thevirus in vivo. Given it is thought to be desirable to establish a viralinfection in a cancer cell such that the virus can replicate, thenco-administration of compounds that inhibit viral replication is likelyto be undesirable.

In one embodiment the chemotherapeutic agent is not an enzyme inhibitor.Thus in one embodiment the combination therapy does not employ atopoisomerase inhibitor.

In one embodiment he chemotherapeutic agent is not a parp inhibitor.

In one embodiment the combination therapy employs a platinum containingchemotherapeutic agent, for example cisplatin, carboplatin oroxaliplatin.

In one embodiment the combination employs a microtubule inhibitor, forexample vincristine sulphate, epothilone A,N-[2-[(4-Hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide(ABT-751), ataxol derived chemotherapeutic agent, for examplepaclitaxel, abraxane, or docetaxel or a combination thereof.

In one embodiment the combination employs an mTor inhibitor. Examples ofmTor inhibitors include: everolimus (RAD001), WYE-354, KU-0063794,papamycin (Sirolimus), Temsirolimus, Deforolimus(MK-8669), AZD8055 andBEZ235(NVP-BEZ235).

In one embodiment the combination employs a Pi3 Kinase inhibitor.Examples of Pi3 kinases inhibitors include: GDC-0941, ZSTK474, PIK-90,LY294002, TG100-115, XL147, GDC-0941, ZSTK474, PIK-90, LY294002,TG100-115, XL147, AS-605240, PIK-293, AZD6482, PIK-93, TGX-221,IC-87114, AS-605240, P1K-293, AZD6482, PIK-93, TGX-221, IC-87114 andcompounds disclosed in WO2011/048111 incorporated herein by referenceincluding2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-(2-(2-methoxyethoxy)ethoxy)prop-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)hex-5-ynoicacid;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;3-((2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-(3-(2-(2-hydroxyethoxy)ethoxy)prop-1-yn-1-yl)-4-oxoquinazolin-3(4H)-yl)methyl)benzonitrile;2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-(2-morpholinoethoxy)prop-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(3-chlorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(3-chlorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(2-fluorobenzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)methyl)-5-ethynyl-3-(2-fluorobenzyl)quinazolin-4 (3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(3-methoxybenzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(3-methoxybenzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(3-(tri-fluoromethyl)benzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(3-(trifluoromethyl)benzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(4-chlorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(4-(methylsulfonyl)benzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(4-(methyl-sulfonyl)benzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(4-(trifluoromethyl)benzyl)quinazolin-4(3H)-one;3-((2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-4-oxo-quinazolin-3(4H)-yl)methyl)benzonitrile;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(3-(methyl-sulfonyl)benzyl)quinazolin-4(3H)-one;3-((2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-4-oxo-quinazolin-3(4H)-yl)methyl)benzonitrile;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(4-chlorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(4-chlorobenzyl)-5-(3-methoxy-prop-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-cl]pyrimidin-1-yl)methyl)-3-(3-methoxybenzyl)-5-(3-methoxyprop-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-methoxyprop-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(4-(trifluoromethyl)benzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-(2-methoxyethoxy)prop-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-((5-methylisoxazol-3-yl)methyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-((5-methylisoxazol-3-yl)methyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(3-chloro-2-fluoro-benzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2,6-difluorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)methyl)-3-(4-chloro-2-fluorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(3-fluoro-4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)methyl)-5-(3-methoxyprop-1-ynyl)-3-(3-(trifluoromethyl)benzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)methyl)-5-ethynyl-3-(4-fluorobenzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-cyclopentylprop-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-(3-(benzyloxy)prop-1-ynyl)-3-(2-chloro-benzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(5-hydroxypent-1-ynyl)quinazolin-4(3H)-one;21(4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(2-fluoro-5-methoxybenzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(3,4-dichlorobenzyl)-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-benzyl-5-ethynylquinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(2-trifluoromethylbenzyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(4-methoxybenzyl)quinazolin-4(3H)-one;4-((2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-4-oxoquinazolin-3(4H)-yl)methyl)benzonitrile;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-ethynyl-3-(2-fluoro-4-methoxybenzyl)quinazolin-4(3H)-one;1-(3-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)prop-2-ynyl)urea;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-fluorobenzyl)-5-(3-(2-(2-methoxyethoxy)ethoxy)prop-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-fluoro-3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-ethynyl-quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(3-phenoxyprop-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)methyl)-3-(2-fluorobenzyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;6-(21(4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(2-methoxyethyl)hex-5-ynamide;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(7-morpholino-7-oxohept-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(5-morpholino-5-oxopent-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-((5-methylpyrazin-2-yl)methyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-oxo-6-(piperidin-1-yl)hex-1-yn-1-yl)quinazolin-4(3H)-one;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-diethylhex-5-ynamide;7-(2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chloro-benzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)hept-6-ynoicacid;2-Acetamido-N-(3-(2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)prop-2-yn-1-yl)acetamide;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(3-methoxy-5-(trifluoromethyl)benzyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-methoxyphenethyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(benzo[b]thiophen-2-ylmethyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-fluoro-3-methoxybenzyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;Methyl3-((2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)-4-oxoquinazolin-3(4H)-yl)methyl)benzoate;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-((1-methyl-1H-pyrazol-4-yl)methyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)methyl)-3-(benzofuran-5-ylmethyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-((2-methylthiazol-4-yl)methyl)-5-(6-morpholino-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-methylpiperazin-1-yl)-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-morpholinopiperidin-1-yl)-6-oxohex-1-yn-1-yl)quinazolin-4(3H)-one;5-(6-(4-Acetylpiperazin-1-yl)-6-oxohex-1-yn-1-yl)-2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)quinazolin-4(3H)-one;N-(4-(2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)but-3-yn-1-yl)morpholine-4-carboxamide;5-(6-(4-Acetyl-piperazin-1-yl)-6-oxohex-1-yn-1-yl)-2-((4-amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)quinazolin-4(3H)-one;N-(4-(2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)but-3-yn-1-yl)morpholine-4-carboxamide;2-((4-Amino-3-(4-hydroxy-phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-(5-(bis(2-methoxyethyl)amino)pent-1-ynyl)-3-(2-chlorobenzyl)quinazolin-4(3H)-one;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-cyclopentylhex-5-ynamide;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(tetrahydro-2H-pyran-4-yl)hex-5-ynamide;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(2-morpholinoethyl)hex-5-ynamide;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-(2-methoxyethyl)piperazin-1-yl)-6-oxohex-1-ynyl)quinazolin-4(3H)-one;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-a]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(2-(dimethylamino)ethyl)hex-5-ynamide;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(pyridin-4-yl)hex-5-ynamide;6-(2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(pyridin-4-yl)hex-5-ynamide;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-(dimethylamino)piperidin-1-yl)-6-oxohex-1-ynyl)quinazolin-4(3H)-one;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide;6-(2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-bis(2-methoxyethyl)hex-5-ynamide;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(2-(4-methylpiperazin-1-yl)ethyl)hex-5-ynamide;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-methyl-N-(2-(4-methylpiperazin-1-yl)ethyl)hex-5-ynamide;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-isopropylhex-5-ynamide;6-(2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-isopropylhex-5-ynamide;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N,N-dimethylhex-5-ynamide;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-oxo-6-(pyrrolidin-1-yl)hex-1-yn-1-yl)quinazolin-4(3H)-one;6-(2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-4-oxo-3,4-dihydroquinazolin-5-yl)-N-(pyrrolidin-3-yl)hex-5-ynamide;21(4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(3-(dimethylamino)pyrrolidin-1-yl)-6-oxohex-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(3-(dimethylamino)pyrrolidin-1-yl)-6-oxohex-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(4-hydroxyphenyl)-1H-pyrazolo[3,4-c]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-methyl-1,4-diazepan-1-yl)-6-oxohex-1-ynyl)quinazolin-4(3H)-one;2-((4-Amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-(4-methyl-1,4-diazepan-1-yl)-6-oxohex-1-ynyl)quinazolin-4(3H)-one,2-((4-Amino-3-(4-hydroxy-3-methoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-3-(2-chlorobenzyl)-5-(6-morpholino-6-oxohex-1-ynyl)quinazolin-4(3H)-one

or a pharmaceutically acceptable salt thereof, including allstereoisomers, tautomers and isotopic derivatives thereof

In one embodiment the combination employs a MEK inhibitor. Examples ofMEK inhibitors include: A5703026, CI-1040 (PD184352), AZD6244(Selumetinib), PD318088, PD0325901, AZD8330, PD98059, U0126-EtOH, BIX02189 or BIX 02188.

In one embodiment the combination employs an AKT inhibitor. Examples ofAKT inhibitors include: MK-2206 and AT7867.

In one embodiment the combination employs an aurora kinase inhibitor.Examples of aurora kinase inhibitors include: Aurora A Inhibitor I,VX-680, AZD1152-HQPA(Barasertib), SNS-314 Mesylate, PHA-680632,ZM-447439, CCT129202 and Hesperadin.

In one embodiment the combination employs a p38 inhibitor, for exampleas disclosed in WO2010/038086, such asN-[4-({4-[3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido]naphthalen-1-yloxy}methyl)pyridin-2-yl]-2-methoxyacetamide.

In one embodiment the combination employs a Bcl-2 inhibitor. Examples ofBcl-2 inhibitors include: obatoclax mesylate, ABT-737,ABT-263(navitoclax) and TW-37.

In one embodiment the combination employs an antimetabolite. Examples ofan antimetabolite include: capecitabine (xeloda), fludarabine phosphate,fludarabine(fludara), decitabine, raltitrexed(tomudex), gemcitabinehydrochloride and cladribine.

In one embodiment the therapeutic agent is ganciclovir, which may assistin controlling immune responses and/or tumour vasculation.

In one embodiment one or more therapies employed in the method hereinare metronomic, that is a continuous or frequent treatment with lowdoses of anticancer drugs, often given concomitant with other methods oftherapy.

Subgroup B oncolytic adenoviruses, in particular Ad11 and those derivedtherefrom such as ColoAd1 may be particularly synergistic withchemotherapeutics because they seem to have a mechanism of action thatis largely independent of apoptosis, killing cancer cells by apredominantly necrolytic mechanism. Moreover, the immunosuppression thatoccurs during chemotherapy may allow the oncolytic virus to functionwith greater efficiency.

In one embodiment the chemotherapeutic agent is administeredparenterally.

In one embodiment the chemotherapeutic agent is administered separatelyto the virus, either temporally or by an alternate method ofadministration or both. Treatment can be concurrent or sequential.

In one embodiment the cancer treatment is a targeted agent, for examplea monoclonal antibody such as bevacizumab, cetuximab or panitumumab orantibody conjugate, such as an antibody drug conjugate, in particular ofthe type where the antibody or binding fragment is linked to a toxin.

In one embodiment the cancer treatment is an immunotherapeutic agent,for example ipilimumab or other anti-CTLA4, anti-PD-1, anti-PD-L1, orother checkpoint inhibitors, or a cytokine or a cytokine analogue.

Checkpoint inhibitor as employed herein is intended to refer to agentsthat inhibit signalling from T-cell membrane proteins that act toinhibit or downregulate T-cell activation and function. In oneembodiment the virus is administered in combination with theadministration of radiotherapy.

Radiotherapy as employed herein is intended to refer to the medical useof ionising radiation.

Cancer cells are generally undifferentiated and stem cell-like; theyreproduce more than most healthy differentiated cells, and have adiminished ability to repair sub-lethal damage. DNA damage is thenpassed on through cell division; damage to the cancer cells' DNAaccumulates, causing them to die or reproduce more slowly.

In one embodiment the radiotherapy is administered concurrently.

In one embodiment the radiotherapy is administered sequentially.

In one embodiment the virus is administered in combination with therapycomplimentary to the cancer therapy, for example a treatment forcachexia, such as cancer cachexia, for example S-pindolol, S-mepindololor S-bopindolol. Suitable doses may be in the range of 2.5 mg to 100 mg,such as 2.5 mg to 50 mg per day provided a single dose or multiple dosesgiven as multiple doses administered during the day.

In one embodiment the virus is administered in combination with theadministration of one or more prophylactic agents, for example selectedfrom an antipyretic, an antihistamine, an antiemetic, an antidiarrheal,steroid and an analgesic.

Antipyretics include aspirin and non-steroidal anti-inflammatories, forexample ibuprofen, naproxen and ketoprofen.

Antihistamines include acrivastine, azalastine, brompheniramine,buclizine, bromodiphenhydramine, carbinoxamine, cetirizine,chlorpromazine, cyclizine, chlorpheniramine, chlorodiphenhydramine,clemastine, cyproheptadine, desloratadine, dexbrompheniramine,deschlorpheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene,diphenhydramine, doxylamine, ebstine, embramine, fexofenadine,levocetirizine, loratadine, meclizine, mirtazapinem olopatadrine,pheninidamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine,quetiapine, rupatadine, tripelennamine and triprolidine.

Antiemetics include dolasetron, granietron, ondansetron, tropisetron,palonoestron, mirtazapine, domperidone, olanzapine, droperidol,metoclopramide, alizapride, prochloperazine. In some instancesantihistamines may be employed as antiemetics.

Antidiarrheals include methylcellulose, attapulgite, bismuthsubsalicylate, atropine/diphenoxylate, loperamide and other opioids suchas codeine and morphine.

Analgesics include non-steriodal anti-inflammatories, paracetamol, cox-2inhibitors, opiates and morphinomimetics, such as morphine, codeine,oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine,tramadol and the like.

In one embodiment viral treatment is employed in combination with acourse of steroids.

Steroids include hydrocortisone, cortisone, prednisone, prednisolone,methylprednisolone, dexamethasone and the like.

Prophylactic as employed herein is intended to refer to preventivemedicine or care, for example consisting of measures taken to prevent orameliorate side effects during or following administration of the virus.

In one embodiment the prophylaxis is administered separately to thevirus, either temporally or by an alternate method of administration orboth. Treatment can be concurrent or sequential.

In one embodiment additional hydration is provided in combination withthe administration of the virus, either concurrently or sequentially.

Additional hydration as employed herein means the patient is suppliedwith fluids beyond those included in the formulation. This may be anyform of suitable liquid, for example, a saline or glucose infusion.

In one embodiment the virus therapy herein is administered incombination with an anti-inflammatory, for example a steroid ornon-steroidal anti-inflammatory.

In one embodiment the virus therapy according to the present disclosureis administered in combination with an anti-pyretic.

In one embodiment the viral treatment is administered in combinationwith hydration therapy, for example intravenous administration offluids, in particular isotonic saline or glucose.

In one embodiment the method is suitable for treating the patient as anoutpatient.

Outpatient as employed herein is a patient who is not hospitalisedduring the treatment phase, but instead comes to a physician's office,clinic or day surgery for treatment.

In one embodiment there is provided a method of treating a patient witha pharmaceutical formulation described herein comprising ColoAd1 saidmethod comprising the steps of intravenously administering to saidpatient: a dose on day 1 followed by, a dose on day 3, and a third doseon day 5.

In one embodiment there is provided a parenteral formulation of areplication capable oncolytic subgroup B adenovirus described herein,for use in treatment, such as a tumour

and/or malignancy and/or cancer treatment by administering: a first doseof said formulation described herein, followed by one or more furthertherapeutic doses thereof

wherein the first dose and further doses are administered within aperiod of 14 days, in particular as described supra.

In one embodiment, there is provided the use of multiple cycles oftreatment with a replication capable oncolytic subgroup B adenovirus. Atreatment cycle is to be interpreted herein as a series of viral dosesadministered to a patient over a relatively short period of time afterwhich the patient's response will be assessed. Treatment cycles can berepeated multiple times provided the risk benefit is determined to be inthe patient's best interests.

In one embodiment, there is provided a method to assess the suitabilityof repeated cycles of treatment with a replication capable oncolyticsubgroup B adenovirus by determining the level of the specific antiviraltitre and comparing it to the pre-treatment titre, such that a titrebelow a certain percentage of the pre-treatment titre will indicate apositive risk benefit profile for retreatment.

In one embodiment there is provided use of parenteral formulation of areplication capable oncolytic subgroup B adenovirus in the manufactureof a medicament for treatment of a tumour and/or malignancy and/orcancer treatment by employing a treatment regimen herein.

In one embodiment the formulation is employed in the treatment orprophylaxis of metastasis.

In one embodiment, there is provided a formulation presented as asterile prefilled and packaged syringe of appropriate dose and volume inorder to circumvent the need for complex and expensive dose preparationunder sterile conditions and using appropriate air handling such asextraction hoods prior to administration to the patient.

In the context of this specification “comprising” is to be interpretedas “including”.

Aspects of the invention comprising certain elements are also intendedto extend to alternative embodiments “consisting” or “consistingessentially” of the relevant elements.

Any positive embodiment or combination thereof described herein may bethe basis of a negative exclusion i.e. a disclaimer.

EXAMPLES

Preclinical Potency and Selectivity

Table 2 shows the IC₅₀ of ColoAd1 on a variety of epithelial cell lines.

Cell Name Cell Type IC50 HT-29¹ Colorectal cancer 0.06 HT-29² Colorectalcancer 0.04 DLD-1¹ Colorectal cancer 0.35 LS1034¹ Colorectal cancer 0.21HCT116¹ Colorectal cancer 0.02 LS174T¹ Colorectal cancer 0.57 SW48¹Colorectal cancer 0.06 SW403¹ Colorectal cancer 1 HepG2² Hepatoma 0.05PC-3¹ Prostate cancer 0.23 DU145¹ Prostate cancer 5 Panc-1¹ Pancreaticcancer 12 MDA231¹ Breast cancer 0.84 OVCAR-3¹ Ovarian cancer 3 A549²Lung cancer 2 HMEC¹ Capillary endothelial 575 HUVE¹ Umbilicalendothelial 50 HUVE² Umbilical endothelial 60 Hepatocytes² Normal livercells 1050 W138² Fibroblast 350 The IC₅₀ of ColoAd1 on a range ofepithelial derived cancer and normal cells. The number of ColoAd1particles required to kill 50% of cell (IC₅₀) was determined in vitrousing a standard 6 day MTS assay. ¹Results performed by Schering AG andpublished in Kuhn et al., 2008. ²Repeat and additional studies performedby the University of Oxford (unpublished).

Table 3 shows the IC₅₀ of ColoAd1 on a variety of non-epithelial celllines.

Cell name Cell type IC50 Colo320DM¹ neuroendocrine 105 501² melanoma 430IG37² melanoma >500 IG39² melanoma 470 U87MG² Glioblastoma >1000 BBA²Glioblastoma >1000 BBB² Glioblastoma >1000 K562² Leukaemia >1000 TheIC₅₀ of ColoAd1 on a range of non-epithelial derived cancers. The numberof particles required to kill 50% of cell (IC₅₀) was determined in vitrousing a standard 5 day MTS assay. ¹Results performed by Schering AG andpublished in Kuhn et al., 2008. ²Repeat and additional studies performedby the University of Oxford (unpublished).

Table 4 shows ColoAd1 replication in a variety of normal non-cancerhuman cell lines.

Successful Re-infection Genomes Genomes % of of HT29 Human Cell Types(cells) % of control (supernatant) control Cells HT29 (+ve control)3.18E+08 100 4.29E+06 100 YES hepatocytes 6.83E+03 0.0021 2.47E+02 0.01No glomerular endothelial cells 4.78E+02 0.0002 1.22E+03 0.03 No dermalmicrovascular cells 1.03E+03 0.0003 1.45E+03 0.03 No cardiacmicrovascular cells 5.36E+02 0.0002 1.29E+03 0.03 No corneal epithelial2.51E+06 0.7889 7.06E+04 1.65 No bronchial epithelial 5.48E+05 0.17222.88E+04 0.67 No renal cortical epithelial 3.68E+04 0.0116 3.67E+03 0.09No mesangial cells 1.18E+03 0.0004 9.71E+02 0.02 No Intestinalmyofibroblasts 9.50E+02 0.0003 1.49E+03 0.03 No ovarian epithelial2.38E+06 0.7479 9.99E+04 2.33 No astrocytes 7.00E+02 0.0002 9.26E+020.02 No aortic smooth muscle Cells 8.65E+02 0.0003 9.42E+02 0.02 Nocardiac myocytes 1.28E+03 0.0004 7.99E+02 0.02 No renal proximal8.48E+05 0.2666 4.46E+03 0.10 No CD34+ 1.59E+03 0.0005 2.84E+03 0.07 NoPBMC 1.25E+03 0.0004 1.02E+03 0.02 No

Human cells growing in monolayers in vitro were exposed to ColoAd1 for72 hours. The total number of ColoAd1 genome copies was then determinedby qPCR. The data are presented as total genome copies and as a %relative to a carcinoma cell positive control (HT29). The ColoAd1materials derived from these normal human cells were then tested forviability on HT29 carcinoma cells. In all cases ColoAd1 material sorecovered could not be shown to replicate in HT29 cells.

Pre-Clinical Circulation Kinetics

ColoAd1 circulation kinetics were obtained in CD-1 mice. Mice (3 pergroup) were administered virus particles via the tail vein andcirculating genomes in whole blood samples were determined byquantitative PCR (qPCR). ColoAd1 half-life in this model isdose-dependent. At the lower input doses (1×10⁹-2×10¹⁰ on multipledosing days), the mean alpha half-life is 1.8+/−0.5 minutes, consistentwith values previously reported for other adenoviruses (Green 2004). Athigher doses (over 2×10¹¹), saturation of clearance appears to occur,giving rise to longer circulation levels (mean alpha half-life 7.8+/−2minutes). Saturation in the ColoAd1 study described here is reflectedvia multiple pharmacokinetic parameters

Table 5 demonstrates significant increases in Area Under the Curve(AUC), half-life and percentage of particles retained at the 30 mintime-point when ColoAd1 doses were administered over 2×10¹¹. It wasnoted in particular that the optimal kinetics (AUC, percentage retainedparticles at 30 min and mean alpha t½) were all achieved when threeequal high doses were administered as opposed to a low priming dosefollowed by higher doses. From this data, given that blood circulationtimes are significantly longer in humans than in mice, it wasanticipated that the half-life in humans would be considerably longerand that low priming doses were unlikely to be of value fora sub-group Badenovirus. In addition, in tumour bearing human patients, it wasanticipated that replication of this virus in cancer cells, withsubsequent release, would also result in further amplification of thevirus at later time points. The clinical studies were plannedaccordingly.

TABLE 5 Circulation kinetics of ColoAd1 following multipleintra-venousinjections in CD-1 mice (3 mice per group) Mean Alpha Mean AUC Mean % ofinput Study Dose t½ (minutes) ml⁻¹ min⁻¹ virus at 30 min multi-dosestudy in CD1  1 × 10⁹ (d1) 2.2 1.04 × 10⁹  0.77 mice 1 × 10⁹ on day 1then 1 × 10¹⁰ (d3) 2.6 9.17 × 10⁹  0.52 1 × 10¹⁰ on days 3 and 5 1 ×10¹⁰ (d5) 2.6 1.20 × 10¹⁰ 0.42 multi-dose study in CD1 1 × 10¹⁰ (d1) 1.21.06 × 10¹⁰ 0.10 mice 1 × 10¹⁰ on day 1 1 × 10¹¹ (d3) 1.3 9.20 × 10¹⁰0.75 then 1 × 10¹¹ on days 3 1 × 10¹¹ (d5) 1.2 1.06 × 10¹¹ 0.97 and 5multi-dose study in CD1 2 × 10¹⁰ (d1) 1.7 1.71 × 10¹⁰ 0.29 mice 2 × 10¹⁰on day 1 2 × 10¹¹ (d3) 3.7 3.00 × 10¹¹ 4.52 then 2 × 10¹¹ on days 3 2 ×10¹¹ (d5) 4.0 6.31 × 10¹¹ 13.62 and 5 multi-dose in CD1 mice 2 × 10¹¹(d1) 6.5 1.09 × 10¹² 27.43 all three doses at 2 × 10¹¹ 2 × 10¹¹ (d3)10.1 1.21 × 10¹² 28.67 on days 1, 3 and 5 2 × 10¹¹ (d5) 6.8 7.875 ×10¹¹  10.48

Pre-Clinical Interaction Studies

Virus particles can interact with components of human blood includingantibodies, complement and blood cells leading to rapid neutralisation(Lyons 2005, Carlisle 2009). These events are species-specific andcannot be modelled effectively in animals.

To evaluate neutralisation in human blood, ColoAd1 may be incubated infreshly isolated whole human blood from individuals before being appliedto permissive cells (HT29 colorectal tumour cells). A range of virusconcentrations can be chosen to cover the target clinical dose range(2×10⁶ to 2×10⁹ particles per ml of human blood, and assuming a range ofhuman blood volumes). Residual virus potency can be determined bycytotoxicity and compared to virus infection in the absence ofincubation in human blood (media alone) and potency levels within aconcentration in the range 2×10⁶ to 2×10⁹ viral particles per ml isdesirable.

The data in FIG. 1 further demonstrate that ColoAd1 was only marginallyaffected by human blood. Fresh human blood was collected from 9 subjects(A-I) using lithium heparin tubes. ColoAd1 virus particles were added tothe blood samples at 10 fold dilutions from 2×10⁹ VP/mL, which reflectsa potential equivalent human dose of 1×10¹³ assuming that the dose isfully diluted in the total blood volume (assumed to be 5 L of blood).After 20 minutes incubation at 37° C., the virus/blood mixture was addedto A549 tumour cells growing in a 96-well plate. The proportion ofviable A549 cells remaining was then determined after 5 days and plottedas a percentage. The IC₅₀ occurs at a level of approximately equivalentto a viral blood concentration of 2×10⁶ VP/mL. This level of virus wasthus determined as a minimum target level to achieve in the humanclinical studies

Several in vitro studies were also conducted of the interaction ofColoAd1 with human blood cells. Fresh blood was obtained from 4individuals, and erythrocytes, platelets and leukocytes were washed andre-suspended in PBS at physiological cell concentrations (5×10⁹, 2×10⁸and 6×10⁶ per mL, respectively) for use in individual experiments. qPCRanalysis revealed that over 80% (82%±8%) of the ColoAd1 was associatedwith human blood cells, primarily to erythrocytes and leukocytes. Therewas no significant difference in the fraction of ColoAd1 bound to bloodcells after a 5 or 30 minutes of incubation. Ad5 showed comparativelyhigher levels of binding to human blood cells (95.5±1.2%) than ColoAd1.Based on relative fluorescence, pre-incubation of ColoAd1-gfp with humanblood cells for 30 minutes significantly inhibited (>90%) the infectionof SW480 tumour cells, which express only low levels of CD46, thecellular receptor for ColoAd1. Infection of HT29 cells, which expresshigher levels of CD46, was inhibited to a much lesser extent (˜41%),probably because of the higher level of expression of the ColoAd1receptor on these tumour cells. Finally, the infection of leukocytes,which may express high levels of CD46 and thus serve as a “sink” forColoAd1, was assessed using ColoAd1-gfp to determine the extent oftransgene expression. After 24 hours no evidence of transgene expressionwas observed in leukocytes. In contrast, previous studies have shownthat Ad5 is able to efficiently infect monocytes in vitro under the sameconditions. In summary, these studies suggest that the interaction ofColoAd1 with cellular blood components is limited and significantlydifferent to that of Ad5. Again, the clinical studies were designedtaking this into account.

Pre-Clinical Biodistribution of ColoAd1

Biodistribution and clearance of ColoAd1 has been determined in normalmice and transgenic mice expressing the primary virus receptor CD46 (areceptor for group B adenoviruses that is not expressed in normal mice).Following tail vein administration of 1×10¹¹ virus particles in normalmice, virus particles were predominantly found in the liver, spleen andlungs after 24 hrs. (FIG. 2) indicates viral copies per mg and so theselarger organs represent the predominant site of total viral distributionon a percentage basis). Similar distribution to the same target organswas observed in CD46 transgenic mice (FIG. 3), showing that the CD46receptor is not a significant determinant of distribution. Thedistribution of a non-replicating mutant (ColoAd1CJ132) was identical tothat of ColoAd1 indicating replication was not responsible for any ofthis distribution effect. However, in tumour bearing human patients, itwas anticipated that replication of this virus in cancer cells, withsubsequent release, would also result in further amplification of thevirus at later time points and so the clinical studies were plannedaccordingly.

Pre-Clinical Viral Clearance

To identify the time to complete virus clearance, a long-term particleclearance study was carried out in normal Balbc mice. The dominantorgans for virus distribution: liver, spleen and lungs, were chosen foranalysis. Here, the total virus particles per organ are recorded as apercentage of the input dose at each time point such that the resultsare not normalised to organ weight. At 1 hour the majority of the inputvirus has already been sequestered in the Liver, with less than 5% inthe spleen and less than 0.1% in the lungs. At 24 hours post injectionvirus particles have rapidly been cleared from these organs with lessthan 1% of the input virus genomes remaining. Beyond day 65post-injection, no significant levels of virus were detectable in anytissues and levels were not significantly above background. No virusparticles could be recovered for any tissues at day 65post-administration. The kinetics of viral clearance (data presented as% of input dose per organ) are summarised in FIG. 4.

CD46 transgenic mice were administered ColoAd1 on a single occasion at adose of 1×10¹⁰ vp/mouse by tail vein. n=3 animals per time-point. Genomecopies (measured by qPCR) are presented as a percentage of the inputdose of genome copies.

Pre-Clinical Immunogenicity

It is possible that the development of a specific anti-viral immuneresponse may significantly impact the circulation kinetics. To examinethis possibility, a group of mice were administered ColoAd1 repeatedlyover several months in order to produce a pool of hyper-immune serum. Asecond group of mice were then passively immunised against ColoAd1 usingthe hyper-immune serum administered by i.v injection of 10 or 20 ul.These mice were then rested for 10 minutes before being administered5×10¹⁰ ColoAd1 i.v. Blood was collected from each mouse at 2, 10 and 30minutes post-injection of ColoAd1 then analysed by qPCR. The results areshown in FIG. 5 and show that an immune response to ColoAd1 will have asignificant impact upon the kinetics and delivery of ColoAd1, thusdemonstrating the importance of administering doses before such aresponse occurs.

Pre-Clinical Safety and Toxicity

Several safety and toxicity studies have been conducted with ColoAd1,including pilot studies in CD-1 and Balb/c mice, CD46 transgenic mice.In a final toxicity study in male and female CD-1 mice, ColoAd1 wasadministered as three doses given over a 5-day period (on Days 1, 3 and5) to model the intended clinical dosing regimen. Male and female CD-1mice received intravenous bolus injections (dose volume=100 μL) ofColoAd1 or the formulation buffer as shown in Table 6, which shows thefinal study design after the unscheduled deaths of two Group 4 males onDay 1 led to the lowering of the dose in that particular group.

TABLE 6 Toxicity Study Design Main Study Recovery Group Satellite GroupGroup No. of Animals No. of Animals No. of Animals Test Total Dosage No.Males Females Males Females Males Females Item (vp/animal) 1 8 8 8 8 9 9 Vehicle — 2 8 8 8 8 9  9 ColoAd1  6.6 × 10⁹ 3 8 8 8 8 9  9 ColoAd1 6.6 × 10¹⁰ 4  2^(a) —  4^(b) — 2^(b) — ColoAd1  2.2 × 10¹¹  6^(c) — 4^(c) — 7^(c) — ColoAd1 3.59 × 10¹¹ — — — — 3^(d) — ColoAd1 2.09 × 10¹¹—  8^(e) — 8^(e) 9^(e)  9^(e) ColoAd1 2.09 × 10¹¹ ^(a)found dead on Day1 after treatment; received a single dose of ColoAd1 on Day 1 at 2.2 ×10¹¹ vp ^(b)deemed unsuitable for further dosing and euthanised on Day2; received a single ColoAd1 dose on Day 1 at 2.2 × 10¹¹ vp ^(c)receiveda single dose of 2.2 × 10¹¹ vp ColoAd1 on Day 1, then subsequent dosesof 6.96 × 10¹⁰ vp on Days 3 and 5 ^(d)additional males added forcytokine assessment received 6.96 × 10¹⁰ vp on Days 1, 3 and 5 ^(e)atthe top dose all females and all satellite group males received 6.96 ×10¹⁰ vp on Days 1, 3 and 5

A standard set of safety endpoints, including clinical signs, bodyweight, plasma cytokine levels, clinical pathology and gross andmicroscopic examinations were done periodically. A standard list oftissues and organs was collected at necropsy on Days 6 and 17.

No significant clinical signs were observed in males and females inGroups 1, 2 or 3 on any treatment day. Clinical signs of adverse effectwere seen in Group 4 after the first dose on Day 1 at doses of both2.2×10¹¹ and 6.96×10¹⁰ vp/animal, but—with the exception of one Group 4male on Day 3—further adverse clinical signs were not seen. Dose-relatedbody weight loss on Day 2 was seen in all ColoAd1-treated groups exceptfor Group 2 males, though body weight was subsequently unaffected in anytreatment group. Haematological and liver function changes, whenrecorded, occurred over a longer time course but had returned to normalrange by the end of the recovery period. In summary, the mostsignificant clinical signs were seen following the first dose, withsubsequent doses being better tolerated.

Cytokine responses over time in this study are shown in FIG. 6.Elevations of the cytokine MCP-1 were most marked and seen in Groups 3and 4 on Day 1 at 6 and 24 hours post-first treatment and 6 hours aftertreatment on Day 5. No consistent elevation in any other cytokine wasseen in Group 2 animals. Smaller, but dose-related, increases in IL-6,IFNγ and TNFα were seen in Group 3 and 4 animals only, most commonly atlow concentrations compared to MCP-1 and often in only some animals ineach dose group, particularly in Group 3.

The cytokine pattern seen in this study is thus consistent with theclinical signs observed, showing that after the first dose, eachsubsequent dose is better tolerated, even though the doses are equal.

Clinical Studies

At the time of filing, two clinical studies are being conducted toexamine the safety and efficacy of ColoAd1 when delivered intravenouslyto human subjects with metastatic cancer.

The Evolve study (ColoAd1-1001) is a phase I/II clinical study with thephase I dose escalation component conducted in patients with anepithelially derived metastatic tumour (of any origin) and who have nofurther treatment options. Patients in this phase I dose escalation partof the study have been dosed with three equal doses of intravenousColoAd1 on days 1, 3 and 5 (48 hours apart). A slow intravenous infusionhas been used, and in the early cohorts, each patient was infused with30 ml of viral suspension over a 5 minute period (6 ml per minute).Initially each cohort of three patients was dosed at one log incrementsstarting at 1×10¹⁰ viral particles per dose until adverse eventssuggested dose limiting toxicity. Each patient also received a regimenof symptomatic prophylaxis, including supplemental fluids and a setregimen of anti-inflammatories (acetaminophen/paracetamol andibuprofen). The safety and tolerability of this dosing regimen at eachdose level was assessed using physical examinations (including bloodpressure, pulse and temperature) and by eliciting all adverse events, aswell as by assessing haematology, biochemistry and cytokine profilechanges. Viral kinetics and excretion were assessed using regular blood,urine, stool and sputum samples. Efficacy was assessed by serial CTimaging according to objective criteria. Later stages of this study willgo on to examine the safety and efficacy of the intravenous MaximumTolerated Dose (MTD) of ColoAd1 in patients with metastatic colorectalcancer.

A second clinical study (ColoAd1-1002) is a phase 0 “window ofopportunity” study to compare intravenous delivery with directintra-tumoural delivery of ColoAd1 in patients with a newly diagnosedprimary (non-metastatic) colorectal tumour. Patients in this study willbe dosed pre-surgically with ColoAd1 and the resected tumours will thenbe examined post-surgically to examine the extent of viral delivery,replication and spread following the two different delivery and dosageregimens. The measures of safety and viral kinetics in this study arebroadly similar to those of ColoAd1-1001.

The phase I dose escalation patients in study ColoAd1-1001 were dosedintravenously with ColoAd1 at dose levels up to and including 1×10¹³viral particles, in 7 patient cohorts (i.e. Cohorts 1 to 7) as shown intable 7 below.

TABLE 7 Dosage regimes for Cohorts 1 to 7 in the phase I dose escalationcomponent of the ColoAd1-1001 clinical study. Dose regime (as repeatedon days 1, 3 and 5) Total number of viral Rate of viral Dose particles(VP) Infusion Infusion particle cohort administered Volume durationInfusion delivery number per dose (ml) (min) rate (VP/minute) Dosetolerability 1 1e10 (1 × 10¹⁰) 30 ml 5 min 6 ml/min  2e9 VP/min Welltolerated 2 1e11 (1 × 10¹¹) 30 ml 5 min 6 ml/min 2e10 VP/min Welltolerated 3 1e12 (1 × 10¹²) 30 ml 5 min 6 ml/min 2e11 VP/min Welltolerated 4 1e13 (1 × 10¹³) 30 ml 5 min 6 ml/min 2e12 VP/min Nottolerated (dose limiting toxicity) 5 3e12 (3 × 10¹²) 30 ml 5 min 6ml/min 6e11 VP/min Well tolerated 6 3e12 (3 × 10¹²) 30 ml 20 min  1.5ml/min  1.5e11 VP/min  Well tolerated 7 6e12 (6 × 10¹²) 30 ml 40 min 0.75 ml/min   1.5e11 VP/min  Well tolerated

The side effect profile of ColoAd1 in this study has included fever, flulike illness, transaminitis, thrombocytopenia, neutropenia, diarrhoeaand vomiting. However, at a dose level of 1×10¹³ viral particles infusedover 5 minutes, the dose was not well tolerated. In particular, twopatients suffered dose limiting toxicities (DLT) including a cytokinemediated acute lung injury at this dose and could not tolerate more thana single dose. One patient required steroids to treat this condition.Patients at this dose level also suffered chills, hypertension, pain,transaminitis, PPT prolongation, and D-dimer increases, although allresolved with time. As a result of these toxic effects at this poorlytolerated dose, the dose of ColoAd1 was reduced and then re-escalatedusing slower infusion rates. Using this strategy, doses of 3×10¹² VPover either 5 (cohort 5) or 20 minutes (cohort 6) and 6×10¹² VP infusedover 40 minutes (cohort 7) were all shown to be well tolerated.

This safety data is preliminary at the time of writing, but issupportive of a very similar profile to that seen in mice. However, somepatients continued to have fever and asthenia into the second weekdespite no ongoing dosing, a phenomenon that is consistent with on-goingviral replication in the human tumours (a phenomenon that would not beseen in non-tumour bearing mice). The final maximum tolerated dose forhumans is thus anticipated to be between 1×10¹² and 1×10¹³ viralparticles administered on days 1, 3 and 5 at an infusion rate of up to6×10¹¹ VP/min and with each patient also receiving prophylacticanti-inflammatory medication and intravenous fluids as per theColoAd1-1001 protocol. The final optimal dose regimen is now the subjectof further confirmatory studies.

Table 8 summarises the key viral pharmacokinetic parameters as measuredwith qPCR for each patient in the phase I dose escalation component ofthe ColoAd1-1001 clinical study. These results were largely consistentwith the pre-clinical data. In summary there was a dose dependent cMAXand AUC, and the average alpha half-life was approximately 18 minutesalthough there was an indication of possible saturation kinetics at thehigher doses studied.

TABLE 8 ColoAd1 Pharmacokinetics for Cohorts 1 to 7 Dose Infusion α-halfEOI whole blood Patient (viral time life viral load (DNA C_(max) (DNAAUC (DNA Cohort number * particles) (min) (min) copies/ml) copies/ml)copies L/min) 1 1 1 × 10¹⁰ 5 28.22 1.75 × 10⁶ 1.75 × 10⁶ 8.08 × 10¹⁰ 2 1× 10¹⁰ 5 nd 1.20 × 10⁶ nd nd 3 1 × 10¹⁰ 5 20.2 9.84 × 10⁵ 9.86 × 10⁵4.08 × 10¹⁰ 2 4 1 × 10¹¹ 5 4.913 1.60 × 10⁷ 1.60 × 10⁷ 3.70 × 10¹¹ 5 1 ×10¹¹ 5 7.218 7.42 × 10⁶ 7.41 × 10⁶ 2.22 × 10¹¹ 6 1 × 10¹¹ 5 6.006 1.73 ×10⁶ 2.67 × 10⁶ 5.54 × 10¹⁰ 3 7 1 × 10¹² 5 26.66 1.08 × 10⁸ 1.20 × 10⁸4.84 × 10¹² 8 1 × 10¹² 5 8.046 1.26 × 10⁸ 1.26 × 10⁸ 3.42 × 10¹² 9 1 ×10¹² 5 6.031 2.18 × 10⁸ 2.18 × 10⁸ 2.98 × 10¹² 4 10 1 × 10¹³ 5 11.7 7.30× 10⁸ 7.26 × 10⁸ 1.95 × 10¹³ 11 1 × 10¹³ 5 7.085 3.27 × 10⁸ 4.96 × 10⁸4.52 × 10¹³ 12 1 × 10¹³ 5 67.42 3.57 × 10⁸ 3.47 × 10⁸ 2.99 × 10¹³ 13 1 ×10¹³ 5 19.86 1.27 × 10⁹ 1.23 × 10⁹ 4.82 × 10¹³ 5 14 3 × 10¹² 5 3.7464.79 × 10⁸ 4.79 × 10⁸ 7.61 × 10¹² 15 3 × 10¹² 5 7.754 1.31 × 10⁸ 1.31 ×10⁸ 2.54 × 10¹² 16 3 × 10¹² 5 11.96 2.06 × 10⁸ 2.04 × 10⁸ 1.12 × 10¹³ 617 3 × 10¹² 20 6.779 1.10 × 10⁸ 1.10 × 10⁸ 6.84 × 10¹² 18 3 × 10¹² 209.062 2.35 × 10⁷ 3.37 × 10⁷ 2.23 × 10¹² 19 3 × 10¹² 20 6.151 2.34 × 10⁸2.34 × 10⁸ 1.12 × 10¹³ 7 20 6 × 10¹² 40 46.54 1.80 × 10⁸ 1.80 × 10⁸ 1.21× 10¹³ 21 6 × 10¹² 40 22.72 5.68 × 10⁷ 5.68 × 10⁷ 4.82 × 10¹² 22 6 ×10¹² 40 51.34 6.16 × 10⁷ 6.37 × 10⁷ 5.56 × 10¹² * an indicative numberfor each patient nd: not determined.

FIG. 7 shows the cytokine pattern observed in the cancer patients dosedin the ColoAd1-1001 clinical study in the initial dose escalation phase(up to and including identification of the dose limiting toxicity). Asin the mouse studies, the inflammatory cytokine response seen in humanspeaks after the first administration of ColoAd1 and then reduces forsubsequent administrations. Interestingly, this initial priming effectof ColoAd1 is not reliably seen at the lower doses but is clearly seenat the higher dose, supporting the assertion that a repeated high doseregimen with equal dose levels may be optimal for the intravenousadministration of subgroup B adenoviruses to human cancer patients.

In particular FIG. 7 shows cytokine levels (μg/L) over time in humancancer patients with metastatic solid epithelial tumours afterintravenous doses of ColoAd1 administered as a 5 minute infusion of 30ml of viral suspension on Days 1, 3 and 5 (dose points indicated byarrows) at four different dose levels (1e10, 1e11, 1e12 and 1e13 viralparticles respectively). Each patient also received prophylacticanti-inflammatory medication and intravenous fluids. Patients at dosesup to and including 1e12 tolerated these doses well, but two out of fourpatients who received the 1e13 dose experienced cytokine mediated doselimiting toxicity and were unable to receive more than a single dose.For the individual patients, raised TNF and gamma interferon levelscorrelated well with tolerability, but raised IL6 did not (data notshown). It was thus determined that rates up to 2e11 viral particles perminute could be regarded as a well-tolerated infusion rate. Panel A:TNF; Panel B: gamma interferon; Panel C: IL-6.

FIG. 8 shows systemic pharmacokinetics of ColoAd1 (Genome copies per mLof blood) in human cancer patients with metastatic solid epithelialtumours. Genome copies measured by qPCR.

In particular, FIG. 8A shows the mean plasma level of the three patientsfrom the ColoAd1-1001 dosed with a well-tolerated dose (1e12 VP perdose) administered at 2e11 VP/min as equal intravenous doses of ColoAd1administered as a 5 minute infusion of 30 ml of viral suspension on Days1, 3 and 5 (dose time points indicated by arrows). The trend towardsincreasing viral concentration peaks with each subsequent dose istypical.

This clearly shows the beneficial effect of the claimed dosing regimenon viral pharmacokinetics, with the peak levels of virus after thesecond and third dose being increasingly higher than the peak levels ofvirus after the first dose. This demonstrates the benefit of occupyingor removing the non-cancerous viral sinks with the earlier doses. Thisdose was well tolerated in these three patients.

FIG. 8B shows the mean initial pharmacokinetics (viral DNA copies/ml)following the first dose of virus at four different dose levels forpatient cohorts 1 to 4 (1×10¹⁰, 1×10¹¹, 1×10¹² and 1×10¹³ viralparticles respectively) of the ColoAd1-1001 clinical study. In each casethe dose is administered over 5 minutes and so the viral infusion rateincreases from 2×10⁹ viral particles per minute at the lowest dose to2×10¹² viral particles per minute at the top dose. At the top two doses,the blood viral concentration remains above the 2×10⁶ viral particlesper mL level for a prolonged period. This is the minimum blood level (aspredicted from preclinical studies and as shown in FIG. 1) to beeffective at establishing an infection within the tumour. For the 1×10¹²dose this target level is achieved for between 1 to 2 hours, whilst forthe 1×10¹³ dose this target level is maintained for over 6 hours.However, the 1×10¹³ viral particle dose administered as 2×10¹² viralparticles per minute was poorly tolerated, with two patients sufferingacute cytokine mediated dose limiting toxicity and so this dose regimenis not optimal.

A dose regimen using a well-tolerated infusion rate (such as 2×10¹¹viral particles per minute or slower) may allow the administration ofdoses up to and possibly higher than 1×10¹³ viral particles. Using thisdata, pharmacokinetic modelling can then be used to show that a dose of1×10¹³ viral particles infused over one hour (1.67×10¹¹ viral particlesper minute) will maintain viral blood levels above the 2×10⁶ targetlevel for three hours or more in most patients.

FIGS. 9A to 9H shows the pharmacokinetics from patients in theColoAd1-1001 clinical trial. Patients were administered the first doseof ColoAd1 and the viral load was then assessed with serial blood drawsusing qPCR. The following treatment regimens were tested:

FIG. 9A: 1e10 (1×10¹⁰) viral particles administered over 5 minutes(Cohort 1); FIG. 9B: 1e11 (1×10¹¹) viral particles administered over 5minutes (Cohort 2); FIG. 9C: 1e12 (1×10¹²) viral particles administeredover 5 minutes (Cohort 3); FIG. 9D: 1e13 (1×10¹³) viral particlesadministered over 5 minutes (Cohort 4); FIG. 9E: 3e12 (3×10¹²) viralparticles administered over 5 minutes (Cohort 5); FIG. 9F: 3e12 (3×10¹²)viral particles administered over 20 minutes (Cohort 6); and FIG. 9G:6e12 (6×10¹²) viral particles administered over 40 minutes (Cohort 7).

Each curve represents the viral blood levels measured for an individualtest subject per unit time prior to receiving ColoAd1 and up to about 6hours following treatment.

Adverse side effects were first observed in patients when viral bloodlevels exceeded a threshold of about 3e8 viral genomes per mL.

Hence, a range of about 3e7 to 3e8 viral genomes per ML was determinedto be an ideal therapeutic range and that a regimen which maintains theviral blood levels within this range for as long as possible wouldmaximise viral blood levels, whilst minimising toxic side effects.

As can be seen from the pharmacokinetic curves, FIG. 9G (6e12 particlesadministered over 40 minutes) shows a particularly suitable profile withthe viral blood levels maintained within the therapeutic range for thelongest.

FIG. 10 shows a comparison between C_(max) levels when the same dose isadministered to patients in the ColoAd1-1001 study as either a slowinfusion or a fast infusion. Cohorts 5 and 6 were both administered atotal dose of 3×10¹² viral particles but for cohort 5 the dose wasinfused over 5 mins (fast infusion) and cohort 6 the dose was infusedover 20 mins (slow infusion). It can be seen that slowing the infusionrate can effectively result in less variation of the C_(Max) and a lowermean C_(Max) level, and so limiting the infusion rate of a Group Badenovirus is thus relevant when higher C_(Max) levels are associatedwith toxicity.

FIG. 11 shows MCP1 levels (μg/L) over time in human cancer patientsafter intravenous doses of ColoAd1 administered on Days 1, 3 and 5 (dosepoints indicated by arrows) in ther ColoAd1-1001 clinical study. Thegraph shows the comparison between the different patient cohorts (1 to7) that were each administered with a different dose regimen shown inTable 7.

Measurements of MCP1 levels (μg/L) were taken at the following timepoints: at 0 hours, 6 hours, 12 hours, 24 hours, 48 hours, 54 hours, 60hours, 72 hours, 96 hours, 102 hours, 108 hours, 120 hours, 168 hoursand 336 hours. This human data reflects a similar pattern to that seenin mice with reducing levels of MCP1 after each dose for every dosetested, thus supporting the specific benefits of the claimed doseregimen.

The cytokine pattern seen in FIG. 12 is consistent with the cytokinepatterns previously observed in the mice studies (see FIG. 6), showingthat after the first dose, each subsequent dose is better tolerated evenwhen each of the 3 doses is equal.

Studies Showing that Infection of Tumours by Type B Adenovirus can beEstablished by Doses of Viral Particles Administered by IV

FIG. 12 shows the replication cycle typical for an adenovirus.Adenovirus structural proteins e.g. Hexon, which makes 90% of the viruscapsid, are only expressed late during infection after replication hasoccurred. The proteins are then transported back to the nucleus forassembly. The nucleus thus has the highest concentration of hexon andother structural proteins during replication. Therefore, nuclear hexonstaining can be used for the quantification of adenovirus and as amarker for cells that have been successfully infected with ColoAd1.

In the ColoAd1-1002 clinical study, patients with primary(non-metastatic) colorectal tumours received ColoAd1 by eitherintratumoural (IT) delivery or intravenous (IV) delivery. In the ITgroup, the virus was administered via a colonoscope at a dose of up to1e8 VP as multiple injections (actual dose was dependent upon the tumoursize). In the IV group the dose was 1e12 VP administered as an infusionover 5 min on days 1, 3 and 5. Then, 7 to 14 days after the first doseof ColoAd1, the primary tumour was resected and was sent forpathological examination including immunohistochemical (IHC) stainingfor ColoAd1 hexon.

Sections of formalin-fixed, paraffin-embedded human tumour samples wereanalyzed for the presence of virus using an anti-hexon antibody(ab8251).

Staining was carried out under using a validated assay with a VentanaBenchmark Ultra. Strong nuclear staining indicates the presence of hexonundergoing capsid assembly.

Isotypes controls were processed at the same time and under the sameconditions.

FIG. 13 shows a transmission EM image of a colorectal cancer cell lineinfected in vitro with ColoAd1.

FIG. 14A shows cell staining images of a tumour sample which has beeninfected with ColoAd1 after intratumoural injection (IT), and thenstained for Hexon. As can be seen, there is substantial nuclear stainingin carcinoma cells whereas there is no nuclear staining in stroma cells.FIG. 14B shows the corresponding isotype control. Together these slidesshow that ColoAd1 infects tumour cells selectively, without infectingnormal cells following direct intratumoural delivery.

FIG. 14C shows a cell staining image of a tumour sample which has beeninfected with ColoAd1 after intravenous (IV) administration, and thenstained for Hexon. As can be seen, there is substantial nuclear stainingin carcinoma cells whereas there is no nuclear staining in stroma cells.FIG. 14D is the corresponding isotype control.

Therefore, these images provide clear evidence that ColoAd1 can beselectively delivered to tumour cells in a manner that is equivalent tointratumoural delivery when using the claimed intravenous dosingregimen.

Example 2 Drug Combination

ColoAd1 virus replication in the presence of 320 clinically approvedcompounds or compounds in development was assessed in the coloncarcinoma cell line, HT-29. HT-29 cell were seeded at a density of 3.0e4cells per well in 96 well plates and incubated at 37° C., 5% CO₂. After4-6 hrs incubation virus and drug compound mixtures prepared in cellmedia were diluted onto the cells to give final doses of 10 ColoAd1virus particles per cell (ppc) and 0.1 μM of drug compound. The cellswere incubated for 18 hrs and then the total virus genomes in the cellswere assessed by qPCR. The relative fold change in ColoAd1 replication,compared to ColoAd1 virus alone, is plotted for all compounds in FIG.15. The inset shows an increase in virus replication after 18 hrs in thepresence of microtubule inhibitors and a decrease in virus replicationin the presence of topoisomerase inhibitors.

The effect of paclitaxel or cisplatin treatment on ColoAd1 efficacy in atumour model was assessed in an IP model of ovarian cancer. SCID micewere implanted with 2.5e6 luciferase-expressing SKOV-3 human ovariancarcinoma cells. Tumour burden was assessed by luciferase expression.Mice were imaged on day 5, on the day before each set of treatments andat least every 5-7 days for the duration of the study. All ColoAd1treatments were carried out using 5e9 virus particles delivered byintra-peritoneal injection and in the combined treatment groups,paclitaxel (0.4 mg) or cisplatin (0.04 mg) was delivered the day aftervirus treatment. Disease progression was assessed by luciferase imagingusing an IVIS imaging system. Images of the relative luminescence inmice dosed via IP injection with either PBS (A), paclitaxel (B), ColoAd1(C) or paclitaxel and ColoAd1 (D) is shown in FIG. 16 and the relativeluminescence tracked over time for each dosing group is shown in FIG.17. The relative luminescence in mice dosed via IP injection with eitherPBS (Group 1), ColoAd1 then cisplatin (Group 2), cisplatin then ColoAd1(Group 3) or Paclitaxel and ColoAd1 (Group 4) are shown in FIG. 18.Dosing schedules are detailed in the FIGS. 17 and 18.

We claim:
 1. A method of treating cancer in a human patient, comprisingintravenously administering to the patient multiple doses of aformulation of a replication competent oncolytic adenovirus of subgroupB in a single treatment cycle, wherein a total dose given in eachadministration is in the range of 1×10¹² to 1×10¹³ viral particles, andwherein the rate of viral particle delivery for each administered doseof adenoviruses is in the range of 2×10¹⁰ to 6×10¹¹ particles perminute, and wherein the adenovirus has a fibre and hexon of wild-typeAd11.
 2. The method of claim 1, wherein the period between each doseadministration is in the range of 6 hours to 72 hours.
 3. The method ofclaim 1, wherein the multiple doses are 2, 3, 4, 5, 6 or 7 doses in asingle treatment cycle.
 4. The method of claim 1, wherein the treatmentcycle is a period of 14 days or less.
 5. The method of claim 1, whereineach dose of adenovirus is administered such that the rate of viralparticle delivery is in the range of 1×10¹¹ to 3×10¹¹ viral particlesper minute.
 6. The method of claim 1, wherein 1×10¹³ viral particles areadministered over a 60 minute period per dose or wherein 6×10¹² viralparticles are administered over a 40 minute period per dose.
 7. Themethod of claim 1, wherein a blood level of the adenovirus afteradministration of a second and optionally subsequent doses reaches ablood level of at least 2×10⁶ viral particles per mL.
 8. The method ofclaim 7, wherein the blood level of viral particles is maintained for 15minutes or greater.
 9. The method of claim 1, wherein the volume of theformulation administered is 100 mL or less.
 10. The method of claim 1,wherein the multiple doses comprise a first dose that is administered onday 1, a second dose that is administered on day 3, and a third dosethat is administered on day 5, and further wherein no dose isadministered on day 2 or on day
 4. 11. The method of claim 1, whereinthe adenovirus is a chimeric adenovirus.
 12. The method of claim 1,wherein the adenovirus is administered in combination with theadministration of an anti-cancer therapy, wherein the anti-cancertherapy is an immunotherapeutic agent, a small molecule inhibitor,radiotherapy, radio-isotope therapy or any combination thereof.
 13. Themethod of claim 1, wherein the adenovirus is administered in combinationwith the administration of one or more prophylactic agents.
 14. Themethod of claim 1 for the treatment of a tumour.
 15. The method of claim1, wherein the first dose in the treatment of a given cycle is a lowerdose than the dose administered in subsequent treatments in the cycle.16. The method of claim 1, wherein the same total dose is given in thefirst and the second administrations.
 17. The method of claim 1, whereinthe same total dose is given for all of the administered doses in thecycle.
 18. The method of claim 1, wherein the administration of themultiple doses of replication competent oncolytic adenovirus seeds viralinfection in the cancer cells.
 19. The method of claim 10, wherein theadenovirus is unconjugated.